lajeunesse mqp 4-25dr
TRANSCRIPT
MQP$BIO$JBD$0021------
Structure/Function,Characterization,of,Kekkon5,---
A-Major-Qualifying-Project-Report--
Submitted-to-the-Faculty-of--
WORCESTER-POLYTECHNIC-INSTITUTE--
In-partial-fulfillment-of-the-requirements-for-the--
Degree-of-Bachelor-of-Science--In--
Biology-and-Biotechnology--By--
___________________________________-Chloe-LaJeunesse-
-April-27,-2017-
----APPROVED:--________________________-Joseph-B.-Duffy,-Ph.-D.-Biology-and-Biotechnology-WPI-Project-Advisor-
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ABSTRACT,,Kekkon5- is- a- transmembrane- protein- found- in- Drosophila- melanogaster- with- an-
extracellular- region-comprised-of- leucine-rich-repeats- (LRR)-and-an- immunoglobulin- (Ig)-
domain- and- an- intracellular- region- containing- conserved- sequences-whose- functions- are-
not- well$understood.- To- further- investigate- the- significance- and- function- of- these-
intracellular-Short-Linear-Motifs-(SLiMs),-their-conservation-was-assayed-in-species-across-
the-phylum-Arthropoda- and-variants- in-which- the- second-and- third-motifs-were-missing-
from-Kekkon5-were-generated-and-assayed-in#vivo.!
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ACKNOWLEDGEMENTS,,,,
I-would- first- like- to- thank-Duff- for-making-this-project-possible.-You-have- inspired-
me-so-much-and-have-been-a-wonderful-advisor-from-the-day-you-accepted-me-into-your-lab-
my-freshman-year.-I-will-treasure-and-carry-all-of-the-advice-and-guidance-you-have-given-
me-into-my-future-as-a-scientist.-Thank-you-for-believing-in-me.-I-would-also-like-to-thank-
Alex-Putnam-for-all-of-your-help-in-the-lab-every-day-and-for-staying-late-to-help-me-with-my-
Western-blot.-I-was-really-scared-it-wasn’t-going-to-work,-but-you-were-calm-about-it,-and-it-
ended-up-working!-Without-you,-my-days-in-lab-would-have-been-a-lot-less-fun.-Thank-you-
Natalie- Farny- for- being- a- wonderful- mentor- throughout- iGEM- and- my- undergraduate-
career-alike.--You,-too,-have-inspired-me-more-than-I-can-say-and-I-am-extremely-grateful-to-
have-had-you-as-a-guide.--And-finally,-thank-you-to-the-extraordinary-faculty-and-staff-of-the-
Department-of-Biology-and-Biotechnology.-I-have-learned-so-much-from-all-of-you-and-I-am-
truly-grateful-for-the-time-I-have-spent-here-with-you.---
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TABLE,OF,CONTENTS-,,,ABSTRACT-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2--ACKNOWLEDGMENTS-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3--TABLE-OF-CONTENTS-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4--INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5--MATERIALS-AND-METHODS-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10--RESULTS-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18--DISCUSSION-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -25--REFERENCES-. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28--APPENDIX-A:-SDM-PRIMER-SEQUENCES-AND-LOCATIONS-. . . . . . . . . . . . . . . . . . . . 30--APPENDIX-B:-ONGOING-TRANSGENIC-F1-CROSSES-IN-DROSOPHILA . . . . . . . . . . . . . 31--APPENDIX-C:-GENE-CONSTRUCTION-CONSTRUCTS . . . . . . . . . . . . . . . . . . . . . . . . . 32---
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INTRODUCTION,-
LIG#Proteins##
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LIGs- are- family- of- single$pass- transmembrane- proteins- that- contain- a- relatively-
unique- combination- of- leucine$rich- repeats- (LRRs)- and- immunoglobulin- (Ig)- domains.-
LRRs- are- a- relatively- common-motif- existing- in-hundreds-of- proteins- from-animal,- plant,-
prokaryotic,-and-even-viral-species.-Functionally,-they-are-commonly-associated-with-innate-
immunity,- mRNA- transport,- apoptosis,- neuronal- development,- due- to- their- ability- to-
recognize- and- bind- proteins- and- other- ligands.- (Ko- &- Kim,- 2007;- Ng- et- al.,- 2010).-
Structurally,-LRRs-are-comprised-of-a-core-repetitive-pattern-of-~-nine-amino-acid-resides-
(L$x$x$L$x$L$x$x$N)- that- can- be- repeated- dozens- of- times- within- one- molecule.- When-
stacked- together,- LRRs- adopt- a- concave,- horseshoe$like- structure.- (Padmanabhan- et- al.,-
2009).-
Like- LRRs,- Ig- domains- are- also- a- highly- common- structure.- They- are- more-
characteristically-involved-in-protein-binding,-and-are-most-commonly-found-in-proteins-of-
the- immune-system-such-as-antibodies-and-T$cell- receptors,-but-have-also-been- found- in-
other-cell$surface-receptors,-matrix-proteins,-and-enzymes-(Bork-et-al.,-1994).-Structurally,-
Ig-domains-consist-of-several-folded-antiparallel-β$sheets-connected-by-hypervariable-loop-
structures- that- lend- immunoglobulin- domains- their- ability- to- bind- a- wide- range- of-
structures-(Berg-et-al.,-2002).--
Though-LRRs-and-Ig$like-domains-individually-are-relatively-ubiquitous-in-proteins-
throughout-many- kingdoms- of- life,- their- combination- is- quite- uncommon- and- restricted-
evolutionarily.-In-the-human-proteome,-only-35-proteins-combine-LRR-and-Ig$like-domains,-
compared-to-over-350-human-proteins-containing-LRRs-and-over-1100-proteins-containing-
Ig$like-domains.-In-Drosophila#melanogaster,-only-nine-LIGs-exist-(Evans,-2006).-While-LIG-
proteins- have- yet- to- be-widely- characterized,- past- research- has- determined- that- certain-
LIGs-act-in-signaling-pathways-and-neuronal-development-(MacLaren-et-al.,-2004;-Mandai-
et-al.,-2009).--
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The#Kekkon#Subfamily#and#Kekkon#5#
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Most- LIGs- present- in-Drosophila# melanogaster- are-members- of- the- Kekkon- (Kek)-
subfamily—a-diagram-of-which-can-be-seen-in-Figure-1.-The-Kek-subfamily-is-comprised-of-
six- structurally- related- proteins- (Kek1$Kek6)- that- contain- seven- LRRs- and- one- Ig$like-
domain- in- their-extracellular- region- (MacLaren-et-al.,-2004).-The-cytoplasmic-domains-of-
the- Kekkon- proteins- are- significantly- more- diverse,- containing- unique- but- sometimes-
overlapping-combinations-of-short-stretches-of-highly-conserved-amino-acid-sequences,-or-
short-linear-motifs-(SLiMs)-(MacLaren-et-al.,-2004;-Davey-et-al.,-2006).-There-are-no-direct-
vertebrate- orthologs- of- the- Kek- proteins,- but- the- AMIGO/Alivin- subfamily- of- LIGS-
discovered- in-vertebrates-discovered- in-2003-contain- seven-LRR-and-one- Ig$like-domain,-
much- like- the- Kekkon- subfamily- (Chen- et- al.,- 2006).-While- the- structures- of- the- Kekkon-
proteins-are-well$known,- the- functions-of-each-protein-are-not-as-well$understood.-While-
roles- in-cellular-adhesion-and-neuronal-development-have-been-proposed-for- the-Kekkon-
subfamily- in- general,- Kek1- is- the- only- member- of- the- Kekkon- subfamily- with- a- well$
characterized-function-to-date,-having-a-defined-role-in-inhibiting-Epidermal-Growth-Factor-
Receptor-(EGFR)-signaling-(MacLaren-et-al.,-2004).--
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-Figure,1:,The,Kekkon,subfamily,of,LIGs.,,
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Structurally,- Kekkon5- is- similar- to- its- other- five- relatives,- with- an- extracellular-
region-comprised-of-seven-LRRs-and-one-Ig$like-domain.-The-intracellular-region-of-Kek5-is-
made-up-of-six-SLiMs-that-appear-to-be-highly-conserved—IC1,-IC2,- IC3,- IC4,- IC5,-and-IC6-
(the- PDZ- binding- domain)- (MacLaren- et- al.,- 2004).- Functional- studies- of- Kek5,- have-
suggested-a-role-as-a-regulator-of-bone-morphogenetic-protein-(BMP)-signaling-(Evans-et-
al.,- 2009).- The- BMP- signaling- pathway- is- present- in- many- species- from- Drosophila- to-
humans-and-participates-in-the-development-and-patterning-of-many-tissues.-Misregulation-
of-this-pathway-can-result-in-the-development-of-kidney-disease,-hypertension,-cancer,-and-
other- diseases- (Little- &-Mullins,- 2006).- In-Drosophila,- BMP- signaling- plays- a- key- role- in-
dorsoventral-and-tissue-patterning-in-the-embryo-and-the-development-of-the-wing-in-the-
adult- fly.-Misexpression- of-Kek5- in- the- imaginal-wing- disc,- a- structure- in- the- larvae- that-
gives-rise-to-the-wing,-results-in-several-defects-associated-with-the-BMP-signaling-pathway,-
such-as-the-absence-of-crossveins-in-the-wing-and-the-formation-of-ectopic-scutellar-bristles-
(Evans,- 2006;- Evans- et- al.,- 2009;-Menon,- 2013).-An- example- of- these-phenotypes- can-be-
seen-in-Figure-2-below.-
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-Figure,2:,Missing,anterior,crossveins,in,the,wing,(top),and,ectopic,scutellar,bristle,formation,
(bottom),upon,Kek5,misexpression,(Menon,,2013).,-
Further- studies- have- shown- that- the-misexpression- of- Kek5- in- the- imaginal-wing-
disc-produces-a-lethal-phenotype-in-which-cells-extrude-from-the-wing-imaginal-disc-and-die-
in- Caspase$dependent- manner.- Other- phenotypes- influenced- by- Kek5- misexpression-
(Figure- 3)- include- Armadillo- upregulation- at- the- adherens- junction- and- a- “large- cell”-
phenotype,-in-which-Kek5$misexpressing-cells-appear-to-be-much-larger-than-normal-cells-
(Menon,-2013).-
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Figure,3:,Panels,A,and,B,represent,Armadillo,staining,in,response,to,wildtype,Kek5,(A),or,Kek5%with,deleted,intracellular,domain,(B).,Armadillo,upregulation,is,seen,in,both,cases,while,the,“large,cell”,phenotype,is,seen,only,in,panel,A.,Panels,C,and,D,represent,light,microscope,images,showing,the,cell,extrusion,phenotype,in,response,to,wildtype,Kek5%(A),or,Kek5%with,deleted,
intracellular,domain,(D),(Menon,,2013).,-
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- While-the- importance-of-the-extracellular-region-of-Kek5-has-been-established,- the-
role-of-the-IC-domain-remains-largely-unclear.-In-order-to-better-understand-the-role-of-the-
cytoplasmic-tail,-variants-in-which-different-combinations-of-the-six-defined-IC-SLiMs-have-
been-deleted-have-been-created-and-expressed-in-Drosophila-(MacLaren-et-al.,-2004;-Evans,-
2006;-Evans-et-al.,-2009;-Menon,-2013).-Misexpression-studies-with- these-variants-reveal-
that-the-PDZ-binding-domain-(IC6)-is-necessary-for-Kek5-stabilization-and-localization,-and-
is-thus- indispensable-for-Kek5-function-(Evans,-2006;-Evans-et-al.,-2009).-With-respect-to-
the- remainder- of- the- intracellular- domain,- prior- work- indicates- it- is- critical- for- some-
processes,-but-not-others-(Evans-et-al.,-2009;-Figure-3).--While-evidence-points-towards-IC1-
being-dispensable-in-terms-of-overall-protein-function,-IC2,-IC3,-IC4,-and-IC5-all-appear-to-be-
significant.-In-particular,-the-second-and-third-SLiMs-of-Kek5,-IC2-and-IC3,-seem-to-affect-the-
formation- of- ectopic- scutellar- bristles,- the- cell- extrusion- and- death- phenotype,- and- the-
large$cell-phenotype-(Menon,-2013).-However,-because-only-deletion-variants-coupling-IC2-
and-IC3-with-each-other-and/or-other-SLiMs-have-been-made,-the-individual-effects-of-IC2-
and-IC3-on-Kek5-function-remain-unclear.--
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MATERIALS,AND,METHODS,#
CLUSTAL#Analysis#
Kek5- and- putative- Kek5- sequences- were- gathered- from-Drosophila# melanogaster-
(fruit- fly),- Anopheles# gambiae- (mosquito),- Pediculus# humanus# corporis- (body- louse),-
Acyrthosiphon# pisum- (pea- aphid),- Tribolium# castaneum# (flour- beetle),- Apis# mellifera-
(honeybee),- two- separate- instances- of- Acromyrmex# echinatior- (new- world- ants),- and-
Daphnia# pulex- (water- flea)- in- FASTA- format.- These- sequences- were- then- aligned- using-
EMBL$EBI’s-(Hinxton,-Cambridgeshire)-Clustal-Omega-software.-Amino-acid-residues-were-
color$coded- based- on- characteristics- such- as- hydrophobicity- and- polarity- for- easier-
recognition-of-shared-properties-between-residues.--
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Generation#of#DNA#Constructs#
The-pENTR$Kek5∆2- and- the-pENTRIKek5∆3- constructs-were- generated- synthesizing-
them-from-a#pENTRIKek5-(full$length)-template-through-site$directed-mutagenesis.-Figure-
4- shows- a- summary- of- this- process,- which- was- conducted- with- NEB’s- Q5- Site$Directed-
Mutagenesis-Kit.-In-two-separate-reactions-for-both-IC2-and-IC3,-8-μL-(approximately-25-ng)-
of-pENTR$Kek5-template-was-combined-with-Q5-Hot-Start-High$Fidelity-2X-Master-Mixed-
(12.5-μL),-distilled-water-(2-μL),-a- forward-primer,-and-a-reverse-primer-(10-μM,-1.25-μL-
each)-up- to-a- final-volume-of-25-μL.-These-primers- (Appendix-A)-were-designed- to- flank-
either- IC2- or- IC3- and- read- in- opposite- directions.- In- the- case- of- Kek5∆2,- only- IC2- was-
deleted.-In-the-case-of-Kek5∆3,#to-be-consistent-with-constructs-that-had-included-deletions-
of-IC3-generated-in-the-past,-the-deletion-spanned-from-the-beginning-of-IC3-to-just-before-
the-start-of-IC4.-The-PCR-reaction-for-both-of-these-constructs-was-run-for-25-cycles-with-an-
annealing-temperature-of-62°C-and-an-extension-time-of-2-minutes-and-45-seconds.-
After- the- PCR- was- complete,- a- kinase,- ligase,- and- DpnI- (KLD)- treatment- was-
prepared- for- each- reaction.-The-kinase- and- ligase- allow- the- ends-of- the-PCR-products- to-
ligate- together,- re$establishing- a- circular- vector.- DpnI- is- a- restriction- enzyme- that- only-
digests-methylated-DNA.-Because-the-original-pENTRIKek5#template-was-synthesized-in-E.#
coli,-the-DNA-has-been-methylated.-The-pENTR$Kek5∆2-and-the-pENTRIKek5∆3-constructs,-in-
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contrast,-were-synthesized-in-a-PCR-reaction-and-thus-have-not-been-methylated.-Because-
of- this,- the- addition- of-DpnI-will- degrade- the- template-DNA-while- preserving- the-newly$
synthesized- pENTR$Kek5∆2- or- pENTRIKek5∆3- constructs,- ensuring- that- the- resulting- DNA-
will-have-no-template-present.--
Once- the-KLD- treatment-was- complete,- the-DNA-was- transformed- into-DH5α-cells-
and-plated-onto-on-LB-agar+kanamycin-plates.-Colonies-were-picked-and-grown-up-in-5-mL-
of- liquid- culture- (LB+kanamycin)- and- miniprepped- using- QIAGEN’S- QIAPREP- Spin-
Miniprep-Kit.-Minipreps-of-selected-clones-were-analyzed-via-gel-electrophoresis-and-clones-
likely-to-be-positive-for-pENTR$Kek5∆2-or-pENTRIKek5∆3-were-sequenced-by-Eton-Bioscience-
Inc.- (Boston,- MA)- and- were- verified- in- lab- by- constructing- contigs- of- the- sequenced-
fragments-of-each-construct-in-Sequencher.--
,
,Figure,4:,Site\directed,mutagenesis,in,the,generation,of,Kek5∆2,and,Kek5∆3,pENTR%constructs.-
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Once-pENTR$Kek5∆2-or-pENTRIKek5∆3-were-determined-to-be-successfully-generated,-
they- needed- to- be- transferred- to- an- expression- vector- by- making- use- of- the- Gateway-
cloning-system.-An-overview-of-this-process-can-be-seen-in-Figure-5.---
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-Figure,5:,LR/BP,reactions,in,the,Gateway,cloning,system,in,relation,to,Kek5,constructs.,
,
To- each- reaction- 1- μL- (approximately- 100- ng)- of-pENTR$Kek5∆2- or-pENTRIKek5∆3-
template,-1-μL-(150-ng)-of-pDEST-vector-(in-this-case,-pUASTaGFP),-6-μL-of-distilled-water,-
and-2-μL-of-Invitrogen-Gateway-LR-clonase-were-added.-The-LR-clonase-contains-integrase-
and-excisionase,-two-recombination-enzymes-from-the-lambda-bacteriophage-that-catalyze-
recombination-between-two-vectors-containing-attL-and-attR-sites.-The-attL-and-attR-sites-
become-attP-and-attB-sites,-respectively,-resulting-in-an-expression-clone-of-Kek5-deletion-
variants- flanked- by- attB- sites.- The- LR- reactions-were- left- to- incubate- at- 25°C- for- 1- hour-
before- 1- μL- (2- μg)- of- Proteinase-K-was- added- to- digest- the- recombination- enzymes- and-
terminate-the-reaction.--
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Once-the-reactions-were-complete,-they-were-again-transformed-into-DH5α-cells-and-
plated-onto-on-LB-agar+ampicillin-plates-to-select-for-the-expression-clone.-Colonies-were-
then- picked- and- grown- in-mL- of- liquid- culture- (LB+ampicillin),- then-miniprepped- using-
QIAGEN’S-QIAPREP-Spin-Miniprep-Kit.-Since-PCR-is-not-involved-in-this-cloning-step-there-is-
little-opportunity-for-replication-errors-or-mutation-in-the-LR-reaction-process.-Therefore,-
only-reactions-for-the-∆2-and-∆3-junctions-and-the-GFP-region-of-each-construct-were-sent-
out- for- sequencing- by- Eton- Bioscience- Inc.- (Boston,- MA).- Because- the- original- Kek5-
sequence- did- not- include- a- stop- codon,- when- translated,- the- Kek5- ∆2- and- ∆3- deletion-
variants-will-be-tagged-with-the-eGFP-sequence-included-in-the-original-pUASTaGFP-vector,-
which-does-include-a-stop-codon.-
Upon-verification-of-the-correct-Kek5∆2-or-Kek5∆3-inserts-in-pUASTaGFP,#midipreps-of-
each- constructs- were- prepared- with- QIAGEN’S- QIAGEN- Plasmid-Midi- Kit- to- ensure- that-
enough-DNA-would-be-generated-to-be-sent-off-for-injection-into-Drosophila#melanogaster-
embryos.,
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Gal4/UAS#System#
The-Gal4/UAS-system-is-a-gene-regulatory-system-used- in-research- for-conducting-
gain-of- function-studies- through-controlled-gene-expression-(Duffy,-2002).-The-Gal4-gene-
encodes- a- protein- capable- of- regulating- the- transcription- of- genes- associated- with- an-
Upstream- Activating- Sequences- (UAS)- by- directly- binding- to- the- UAS- to- activate-
transcription.-Originally-discovered-in-the-yeast-Saccharomyces#cerevisiae,- this-system-has-
been- adapted- for- use- in- many- organisms.- For- organismal- gain- of- function- studies- in-
Drosophila,# this- system- is- employed- through- the- crossing-of-driver- (Gal4)-and- responder-
(UAS)- lines.- Lines- of- transgenic- flies- called- responders- that- contain- the- gene- of- interest-
associated-with-UAS-elements-possess-this-gene,-but-do-not-express-it,-as-no-Gal4-is-present-
to- activate- transcription.-These- responders- are-mated- to- lines- of- flies- expressing-Gal4- in-
different- tissues-and-patterns,- the-drivers.-This-results- in-progeny-expressing- the-gene-of-
interest- in- the- tissues- associated- with- the- expression- of- Gal4- in- the- driver- lines- (Duffy,-
2002).- An- overview- of- this- system- is- featured- in- Figure- 6.- This- system- allows- for- the-
misexpression- of- genes- in-Drosophila# (and- other- eukaryotes)-while- also- allowing- for- the-
maintenance-of-a-potentially-lethal-or-otherwise-harmful-gene-in-a-line.--In-cell-culture,-the-
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Gal4/UAS- system- is- also- used- for- gain- of- function- studies- through- the- co$transfection- of-
driver-and-responder-sequences-directly- into- the-culture- (Klueg-et-al.-2002).-The-drivers-
used- in- these- cases- can- be- constitutively- expressed,- such- as- the- ArmIGal4# driver,- or-
inducible,- such- as- the- mtIGal4# driver,- for- which- expression- can- be- induced- by- the-
introduction-of-copper-or-other-heavy-metals-(Klueg,-et-al.,-2002).--
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#Figure,6:,The,Gal4,misexpression,system,in,Drosophila%melanogaster,(Duffy,,2002).-
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Generation#of#Transgenics#
50-μG-each-of-pUASTaKek5∆2GFP-and-pUASTaKek5∆3GFP#DNA-was-sent-to-Best-Gene,-
Inc.- (Chino- Hills,- CA)- for- injection- into- w1118- embryos.- After- injection,- the- larvae- were-
mailed- back- to-Duffy- lab- for- transgenic- screening.- Once- the- larvae- eclosed,- the- resulting-
flies-(generation-G0)-were-single$pair-mated-to-2$3-w1118-males-or-females.-The-pUASTaGFP-
vector- the-Kek5∆2- and-Kek5∆3- genes-were- transposed- into- contained- a- copy- of- the-white-
gene-under-its-own-set-of-regulatory-elements.-Therefore,-any-F1-progeny-from-the-G0-cross-
with- eye- color- are- likely- to- contain- the- transgene.- The- F1- offspring- of- the- G0- cross-were-
screened- for- eye- color,- and- any- putative- transgenics- gathered- from- separate- G0- crosses-
were- considered- independent- insertion- events.- Putative- transgenic- offspring- (generation-
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F1)- for- each- line- were- single$pair- mated- to- 2$3-w1118- males- or- females.- This- mating- of-
putative- transgenics- to-w1118- ensures- the- amplification- of- all- transgenic- lines- for- stable-
stock-generation-and-future-gene-mapping.-
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Transfection#in#Cell#Culture##
# To- confirm- Kek5∆2GFP- and- Kek5∆3GFP- expression- prior- to- the- generation- of- the-
transgenics,-pUASTaKek5∆2GFP-and-pUASTaKek5∆3GFP#were-transfected-into-S3-cells,-a-cell-
line-derived- from-male-Drosophila#melanogaster- embryonic- cells.- First,- each-DNA-sample-
was-purified-using-a-phenol:chloroform-extraction.-An-equal-volume-of-phenol:chloroform-
was- added- to- a- 20- μL- aliquot- of- each- sample- and- after- vortexting,- the- samples- were-
centrifuged-for-2-minutes-at-room-temperature.-To-each-tube,-5-M-NaCl-(in-a-ratio-of-1:20)-
and-100%-EtOH-(in-a-ratio-of-2:1)-were-added-to-precipitate-the-DNA.-The-solutions-were-
incubated-on-ice-for-10-minutes,-then-centrifuged-to-pellet-the-DNA.-Then,-the-DNA-pellet-
was-washed-with-70%-EtOH,-centrifuged-again,-decanted,-and-left-to-air-dry.-Once-dry,-the-
pellets-were-taken-up-in-TE-buffer.-The-concentration-of-each-sample-was-measured-on-a-
NanoDrop,-and-each-construct-was-diluted-to-a-concentration-of-100-ng/µL.-
- Prior- to- transfection,- S3- cells- were- split- into- a- T75- flask- maintained- at- normal-
growth-conditions-(25°C,-no-CO2).-One-day-before-transfection,-on-the-day-the-cells-would-
normally- be- split,- the- S3- cells- in- the- T75- were- resuspended- in- Schneider’s- Media- and-
transferred- to-a-15-mL-conical- tube.-An-aliquot-of- cells-was- taken- from-this- tube,-diluted-
1:10,-and-counted-on-a-hemocytometer.-Then,-according-to-the-appropriate-dilution-factor,-
5-x-106-cells-were-seeded-into-each-well-of-a-6-well-cell-culture-plate-at-a-final-volume-of-1.6-
mL-per-well.-The-plates-were- incubated-at-normal-growth-conditions-for-24-hours-until-a-
confluence-of-approximately-80$100%-was-reached.-
- The-next-day,-four-wells-were-co$transfected-with-400-ng-of-ArmIGal4-(a-constitutive-
driver)- and- 400- ng- of- either-pUASTaKek5wtGFP,# pUASTaKek5∆2GFP,-pUASTaKek5∆3GFP,- or-
nothing-(negative-control)-in-EC-buffer.-Once-Enhancer-and-Effectine-Transfection-reagent-
were-added-and-incubated-in-sequence,-the-media-of-each-well-was-aspirated-and-each-well-
was-washed-with-1X-PBS,-which-was- then-aspirated,-as-well.-1.6-mL-and-0.6-mL-of- fresh-
Schneider’s-Media-was-added-to-each-well-and-each-co$transfection-mix,-respectively.-Each-
16-
!
co$transfection-mix-was-added-to-an-appropriately-labelled-well-and-the-plates-were-left-to-
incubate-at-normal-growth-conditions-overnight.-
#
GFP#Imaging-
Because- the- Kek5# constructs- were- tagged- with- GFP,- the- wild- type- and- variant-
proteins-produced-should-be-visible-through-fluorescent-microscopy.-In-order-to-visualize-
the-Kek5$GFP-proteins-and-observe-expression-and-localization-patterns,-the-co$transfected-
plate-was-viewed-under-under-Zeiss-SteREO-Discovery.V12-microscope-at-100X-either-at-
480- nm- (GFP- fluorescence)- or- under- incandescent- light- (cell- images).- Images- were-
captured-with-a-monochrome-Zeiss-Axiocam-and-processed-with-the-Axiovision-software.--
-
Trypan#Blue#Vital#Stain##
At-day-6-post$transfection,-an-aliquot-of-100-μL-of-resuspended-cells-was-taken-from-
each-co$transfection-well-and-combined-with-100-μL-of-Trypan-blue,-which-is-a-stain-only-
taken- up- by- dead- cells.- For- each- co$transfection,- 10- μL- of- this- Trypan- blue-mixture-was-
loaded-into-a-hemocytometer-and-the-numbers-of-living-and-dead-cells-were-counted-in-two-
separate-squares-and-totaled.-The-percentage-of-dead-cells-to-the-total-number-of-cells-for-
each-co$transfection-was-then-calculated.-
#
Western#Blot#
In-order-to-detect-potential-Kek5wtGFP,-Kek5∆2GFP,-and-Kek5∆3GFP-expression,-125-
μL-(approximately-2-x-107-cells)-of-each-co$transfection-was-aliquoted,-spun-down,- taken-
up- in-1-mL-of- sample-buffer- (12-mM-TrisCl,-5%-glycerol,-0.4%-SDS,-2.9-mM-βME,-0.02%-
bromophenol-blue),-and-stored-at-$20°C.-Just-before-loading,-two-aliquots-of-5-μL-and-20-μL-
for- each- sample- were- made- and- lysed- by- boiling- for- 5- minutes.- Samples- were- run- on-
precast- 7.5%-polyacrylamide- gels- from-BIORAD-at- 20mA- for- 1- hour.- Proteins-were- then-
transferred-from-the-gel-to-a-nitrocellulose-membrane-100V-for-1-hour.-In-order-to-confirm-
the- transfer,- the- membrane- was- Ponceau- stained,- then- blocked- for- 1- hour.- Then,- the-
membrane-was-blocked-in-5%-NFDM-in-TBST-(100-mM-Tris,-150-mM-NaCl,-0.1%-Tween$
20)- for- 1- hour,- after- which- the- membrane- was- rinsed- and- incubated- with- the- primary-
antibody-(monoclonal-α$GFP)-diluted-in-0.5%-NFDM-in-TBST-overnight-at-4°C.--
17-
!
The-next-day,-the-membrane-was-washed-in-TBST-and-incubated-with-the-secondary-
antibody- (goat- α$mouse,- diluted- 1:20,000- in- 5%- NFDM- in- TBST)- for- one- hour- at- room-
temperature.- The- membrane- was- once- again- washed- in- TBST- and- incubated- in- HRP-
substrate-for-10-minutes,-then-photographed-with-chemiluminescent-imaging.,
-
% %
18-
!
RESULTS##
There- is-much- evidence- to- support- links- between- the- IC- domain- of- Kek5- and- its-
overall- function.- Phylogenetic- analysis- reveals- that- all- SLiMs- found- in- the- IC- domain- of-
Kek5-show-a-relatively-high-level-of-conservation-in-closely-related-species,-suggesting-an-
evolutionary-advantage-to-maintaining-these-sequences-(MacLaren-et-al.,-2004;-Talwar-and-
Duffy,- personal- communication).- Additionally,- past- research- has- shown- that- both- the- IC-
domain-as-a-whole-and-certain-combinations-of-IC-SLiMs-have-large-effects-on-the-function-
of-Kek5-(Evans,-2006;-Menon,-2013).-Among-these-combinations,-IC2-and-IC3-in-particular-
have- been- shown- to- have- a- significant- effect- on- phenotypes- associated- with- Kek5-
misexpression-when-deleted-alongside- IC1.-However,-variants- in-which- IC2-and- IC3-have-
been- individually- deleted- have- not- yet- been- successfully- generated.- Therefore,- a- further-
investigation- on- the- both- the- significance- of- these- domains- and- their- individual-
contributions-to-Kek5-function-was-undertaken.--
#
Phylogenetic#Analysis#of#IC2#and#IC3#Conservation#in#Kekkon5#Homologs-
An- alignment- of- Kek5- orthologs- and- putative- orthologs-was- created- using- Clustal-
Omega.-A-sampling-of-Kek5-protein-sequences- for-closely$related-and- less-closely$related-
members- of- Hexapoda- were- analyzed- as- well- as- one-member- of- Crustacea.- The- species-
sampled- for- this-alignment-were,- in-order-of- sequence- in-Figures-7-and-8,-Daphnia#pulex-
(water- flea),-Anopheles#gambiae- (mosquito),-Drosophila#melanogaster- (fruit- fly),-Pediculus#
humanus# corporis- (body- louse),- Acyrthosiphon# pisum- (pea- aphid),- Tribolium# castaneum#
(flour- beetle),- Apis# mellifera- (honeybee),- and- two- separate- instances- of- Acromyrmex#
echinatior-(new-world-ants).--
Figure- 7- shows- the-portion- of- the- alignment- featuring- the- SLiM- $- IC2- (fingerprint-
PDLL).- The- alignment- of- IC2- reveals- that- sequences- highly- similar- to- Drosophila#
melanogaster’s-Kek5-IC2-exist-in-all-sampled-sequences.-
-
-
19-
!
-Figure,7:,Clustal,Omega,alignments,of,Kek5,IC2,in,representative,species,of,Crustacea,(Dpulk5/6),,
and,Chelicerata,(MoKek5),,and,Hexapoda,(all,others).,-
Likewise,- Figure- 8- shows- the- portion- of- the- alignment- featuring- SLiM- $- IC3-
(fingerprint-TIPR).-This-alignment-found-that-sequences-closely-related-in-sequence-to-the-
Kek5-IC3-region-of-Drosophila#melanogaster-exist-in-all-of-the-aligned-sequences.-,
-
-Figure,8:,Clustal,Omega,alignments,of,Kek5,IC3,in,representative,species,of,Crustacea,(Dpulk5/6),,
and,Chelicerata,(MoKek5),,and,Hexapoda,(all,others).,,-
#
Design#of#Novel#Kek5∆2!and$Kek5∆3!Constructs#
Previously,-four-variants-of-Kek5-in-which-IC2-and-IC3-were-deleted-in-combination-
with-other-SLiMs-have-been-generated.-These-constructs-yielded-high-rates-of-lethality-and-
additional- phenotypic- effects- (i.e.- extrusion,- cell- death,- large- cell- phenotype)- when-
expressed- in- Drosophila# melanogaster- (Menon,- 2013).- To- form- a- better- idea- of- the-
individual-effects-and-contributions-of-IC2-and-IC3,-constructs-of-Kek5∆2-and-Kek5∆3-were-
first-synthesized-in-a-pENTR-vector-via-site$directed-mutagenesis-of-an-existing-full-length-
pENTRIKek5-construct.-Then,-Kek5∆2-and-Kek5∆3-were-shuttled-from-the-entry-vector-into-
20-
!
the- expression- vector- pUASTaGFP.- Figure- 9- shows- the- structure- of- the- Kek5- gene- for-
wildtype,-Kek5∆2,-and-Kek5∆3#with-their-respective-lengths.-
-
-Figure,9:,Wildtype,Kek5,and,novel,Kek5,variants:,Kek5∆2,and,Kek5∆3.,
#
Both-the-pENTR-and-pUASTaGFP-constructs-for-each-deletion-variant-were-sequence-
verified.-The-pENTR#constructs-for-Kek5∆2-and-Kek5∆3-were-fully-sequenced-from-the-start-
codon-through-the-end-of-the-gene-to-verify-that-no-mutations-had-accumulated-during-the-
polymerase- chain- reaction.-Because- there- is- little- risk- of-mutation-upon- shuttling- a- gene-
from-one-P$element- vector- to- another- via- the-Gateway- cloning- system,- only- the-deletion-
junction- and- terminal- region- of- pUASTaKek5∆2GFP- and- pUASTaKek5∆3GFP- needed- to- be-
sequenced-to-sufficiently-verify-the-final-constructs.-
-
Generation#of#Transgenic#Lines-
Larvae- injected- with- pUASTaKek5∆2GFP- and- pUASTaKek5∆3GFP# constructs- were-
received-after-mailing-DNA-off- for- injection.- In- total,-143-adult- flies-(Generation-G0)-were-
received- from- these- injections—48- injected-with-pUASTaKek5∆2GFP#and-95- injected-with-
pUASTaKek5∆3GFP.#Each-of-these-flies-were-single$pair-mated-to-2$3-W1118-males-or-females.-
From-these-crosses,-7-putative-Kek5∆2#and-9#putative#Kek5∆3#F1-transgenics-were-collected.-
These-putative-transgenics-have-been-crossed-to-2$3-W1118-males-or-females,-depending-on-
the-sex-of-the-transgenic.--
Figure-10-shows-a-summary-of-the-overall-plan-and-current-state-of-the-generation-
of-transgenic-Kek5∆2#and-Kek5∆3#lines.-These-lines-have-yet-to-be-mapped-and-stabilized,-but-
LRR Ig Tm 1 2 4 5 PDZ 3
LRR Ig Tm 1 4 5 PDZ 3
LRR Ig Tm 1 2 4 5 PDZ
Kek5 wt 2751 bp
Kek5∆2
2664 bp
Kek5∆3
2580 bp
21-
!
the- process- of- doing- so- for- each- line- has- already- begun.- After- the- lines- are-mapped- and-
stable-stocks-are-produced,-functional-tests-can-be-begin-by-crossing-the-Kek5∆2#and-Kek5∆3#
lines-to-lines-expressing-a-Gal4-driver.--
-Figure,10:,Flowchart,of,the,plan,and,progress,for,generating,and,testing,stable,stocks,of,Kek5∆2,and,
Kek5∆3,deletion,variants,in,Drosophila%melanogaster.%,--
-
Expression#and#Localization#of#Kek5#Variants#in#Cell#Culture#
- In- order- to- confirm- the- expression- and- localization- of- the- two- deletion- variants,-
pUASTaKek5∆2GFP-and-pUASTaKek5∆3GFP,-these-constructs-were-co$transfected-into-an-S3-
cell- line- with- a- constitutive- ArmIGal4- driver.- At- 2,- 5,- and- 6- days- post$transfection,-
fluorescent- and- bright- field- microscopy- images- were- taken- of- each- of- the- two- deletion-
variant- constructs- and- positive- (ArmIGal4- +- pUASaKek5wtGFP)- and- negative- (ArmIGal4-
alone)-transfection-controls.-These-images-have-been-compiled-into-Figure-11-below.--
Generate Constructs (2)
Functional Tests
Design Variants
Create Transgenics (48 Kek5∆2 crosses, 95 Kek5∆3 crosses)
Map & Create Stable Stocks (all lines)
Localization/Expression Studies (GFP-based)
Misexpression Studies (A9-Gal4 & Ptc-Gal4 by all lines)
22-
!
-
Figure,11:,Fluorescent,microscopy,of,Arm9Gal4,+,UAS9Kek5wtGFP,,UAS9Kek5∆2GFP,,and,UAS9Kek5∆3GFP,co\transfections,and,Arm9Gal4,control,on,day,2,,day,5,,and,day,6,after,transfection.,Light,microscope,
images,are,displayed,in,each,corner,inset.,-
23-
!
- On-day-2-post$transfection,-the-UASIKek5wtGFP,#UASIKek5∆2GFP,-and-UASIKek5∆3GFP-
ArmIGal4-co$transfections-all-display-GFP-that-appears-to-be-localized-to-the-membrane-of-
each-cell-consistent-with-the-localization-and-expression-pattern-of-Kek5.-As-expected,-the-
control,-which-has-been-transfected-with-only- the-ArmIGal4-driver,-does-not-display-GFP.-
On-day-5-post$transfection,-the-UASIKek5wtGFP-and-UASIKek5∆2GFP-transfections-display-a-
similar- pattern- with- an- apparent- increase- in- levels- and- proportion- of- cells.- However,-
surprisingly-starting-at-day-5-post$transfection,- the-UASIKek5∆2GFP- co$transfection-ceases-
to-display-a-GFP-signal.-This-same-pattern-is-seen-on-day-6-post$transfection.-
#
Cytotoxic#Effect#of#Kek5∆2-#
The- data- gathered- from- the- GFP- localization- and- expression- analysis- in- the-
transfections-revealed-a-decrease-in-GFP-expression-starting-at-day-5-post$transfection-for-
the-ArmIGal4-+-UASIKek5∆2GFP-co$transfection.-Based-on-this-data,-it-was-hypothesized-that-
the-cells-in-the-ArmIGal4-+-UASIKek5∆2GFP#co$transfection-were-dying.-In-order-to-test-this,-
a-vital-stain-(trypan-blue)-was-conducted-using-aliquots-of-cells- from-each-transfection-at-
day- 6- post$transfection.- - A- bar- graph- of- the- percentages- of- dead- cells- observed- for- each-
transfection-can-be-seen-in-Figure-12.--
-
,Figure,12:,Percentage,of,dead,cells,in,trypan,blue,cell,counts,of,Arm\Gal4,+,UAS\Kek5wtGFP,,UAS\Kek5∆2GFP,,and,UAS\Kek5∆3GFP,co\transfections,and,Arm\Gal4,control,at,day,6,after,transfection.,
0%
10%
20%
30%
40%
50%
60%
UAS Kek5wt GFP! UAS Kek5Δ2 GFP!
UAS Kek5Δ3 GFP!
Arm-Gal4!
% c
ell d
eath!
Effect of IC Variants on Cell Viability!
24-
!
The- counts- of- dead- versus- living- cells- in- each- co$transfection- aliquot- reveal- a-
percentage-of- cell-death- that- is-dramatically- increased- in- the-UASIKek5∆2GFP- transfection-
relative-to-the-other-three-transfections.-The-percentage-of-dead-cells-observed-in-the-UASI
Kek5wtGFP,-UASIKek5∆3GFP,-and-the-Arm$Gal4-transfections-is-consistently-close-to-or-below-
10%.- In- contrast,- the- percentage- of- dead- cells- seen- in- the- UASIKek5∆2GFP- transfection-
counts-is-nearly-50%,-suggesting-a-higher-rate-of-death-in-cells-expressing-Kek5∆2GFP.-
-
Analysis#of#Kek5#Variant#Protein#Expression-
In-another-attempt-to-confirm-the-expression-of-Kek5∆2GFP-and-Kek5∆3GFP,-an-anti$
GFP-western-blot-was-run-with-cell-lysates-at-day-6-post$transfection-from-each-of-the-four-
co$transfections.- Figure- 13- is- an- image- of- the- resulting- blot- with- lanes- labelled- and- the-
expected-sizes-Kek5wtGFP-and-UAS$Kek5∆3GFP-listed.--
,Figure,13:,Anti\GFP,Western,blots,of,Arm\Gal4,+,UAS\Kek5wtGFP,,UAS\Kek5∆2GFP,,and,UAS\Kek5∆3GFP,
co\transfections.,-
Western-blot-showed-that-Kek5wtGFP-(lane-1)- is-running-close-to- its-expected-size-
(130-kDa)-and-that-UAS$Kek5∆3GFP-(lane-3)-is-running-slightly-below-at-what-seems-to-be-
its-expected-size-(124-kDa).-UAS$Kek5∆2GFP-did-not-appear-on-the-western-blot.-However,-
given- that- these- cell- lysates- were- taken- at- day- 6- post$transfection,- a- time- point- at- cells-
expressing- UAS$Kek5∆2GFP-were- undergoing- death- and- had- already- ceased- giving- off- a-
GFP-signal,-the-absence-of-a-signal-for-the-∆2-variant-was-expected.-
,, ,
UAS-Kek5
Δ3 GFP
UAS-Kek5
Δ2 GFP
UAS-Kek5
wt GFP
124 kDa
130-kDa
25-
!
DISCUSSION,#
If- cells- cannot- communicate- with- one- another,- than- a- cohesive- multicellular-
organism- cannot- develop.- Cell- signaling- is- a- crucial- aspect- of- the- development- of- all-
animals,-and-while-there-are-many-pathways-throughout-all-existing-species,-many-of-these-
pathways- are- highly- conserved.- One- such- highly$conserved- pathway- is- the- Bone-
Morphogenetic-Protein-(BMP)-pathway,-which-influences-the-patterning-and-development-
of-many-different- types- of- tissues- from-Drosophila# to- humans.-Dysregulation- of- the-BMP-
pathway- can- result- in- the- generation- of- tumors- and- other- diseases- in- humans- (Little- &-
Mullins,-2006).-Because-the-BMP-signaling-pathway-is-widely-conserved,-the-more-insight-
that-can-be-gathered-on-this-pathway-and-how-it-is-regulated,-the-better.--
In- the- past,- the- intracellular- region- of- Kek5- has- been- demonstrated- to- play- some-
role-in-regulating-BMP-signaling-and-to-influence-certain-lethal-cell-phenotypes.-The-second-
and- third-SLiMs-of- the- intercellular- region-of-Kek5- in-particular,- IC2-and- IC3,- seem-to-be-
important-in-regulating-some-aspects-of-BMP-signaling,-as-their-deletion-in-misexpression-
studies-seems-to-influence-a-higher-rate-of-scutellar-bristle-duplication-in-adult-flies-and-IC2-
and-IC3-have-also-been-shown-to-be-key-in-the-development-of-the-lethal-cell-extrusion-and-
large- cell-phenotypes-observed-upon-Kek5-misexpression- in- the-3rd- instar-wing- imaginal-
disc-(Menon,-2013).-Despite-their-apparent-significance-and-the-large-array-of-Kek5-IC-SLiM-
deletion-variants-that-have-been-generated-in-the-past,-their-respective-roles-are-unknown,-
as-individual-deletion-variants-have-not-yet-been-generated.-Therefore,-in-order-to-allow-for-
the-determination-the-significance-and-specific-functions-of-IC2-and-IC3,-a-small-analysis-of-
IC2- and- IC3- conservation- was- undertaken- and- individual- deletion- variants,- Kek5∆2- and-
Kek5∆3,-were-generated.-
- The-results-of-the-phylogenetic-analysis-revealed-that,-within-Hexapoda,-sequences-
representative-of-IC2-and-IC3-are-highly-conserved-within-Kek5-homologs.-Even-in-Daphnia#
pulex,-a-member-of-Crustacea-separated-evolutionarily-from-Hexapoda-by-millions-of-years,-
IC2-and-IC3-are-still-present-within-this-species’-homolog-of-Kek5.-This-large-time-span-of-
conservation-suggests-that,-whatever-their-function-may-be,-IC2-and-IC3-are-indispensable-
for-Kek5-function.-However,-while-Kek5-orthologs-were-searched-for-in-other-members-of-
26-
!
Crustacea- and- members- of- Chelicerata,- there- is- not- much- data- on- orthologous- protein-
sequences-in-general-for-these-species.-In-the-future,-this-study-may-produce-results-more-
representative- of- Kek5- IC2- and- IC3- conservation- in- Arthropoda- species- outside- of-
Hexapoda.--
- Based-on- the- sequencing- information- for- each- construct,- and- the- expression-data,-
the- generation- of- expressible- Kek5∆2# and# Kek5∆3- constructs- was- successful.- While- co$
transfected- cells- expressing-Kek5∆2-exhibited- increased-death-by-day-5-post$transfection,-
the-microscopy-images-taken-on-day-2-post$transfection-suggest-that-Kek5∆2$GFP-is-being-
expressed- and- is- localizing- to- the- cell-membrane-much- like-Kek5wt$GFP-and-Kek5∆3$GFP.-
However,-a-western-blot-of-Kek5∆2$GFP-at-earlier-times-points-have-yet-to-be-performed.-In-
a- future-experiment,- a-new-co$transfection-of-ArmIGal4#and-pUASTaKek5∆2GFP- should-be-
set- up,- and- the- cells- from- that- co$transfection- should- be- harvested- around- day- 2- post$
transfection-for-use-in-a-Western-blot-to-confirm-expression-and-the-size-of-this-protein.-At-
the- same- time,- a-more- closely-monitored- time- course- should- be- taken- of- the-Kek5∆2- co$
transfection-to-gather-more-information-on-the-transition-from-cells-expressing-Kek5∆2$GFP-
to-cells-dying-off.-In-this-way,-it-can-be-determined-whether-or-not-the-observed-cell-death-is-
linked-to-the-expression-of-Kek5∆2$GFP-and-how-this-cell-death-is-occurring.--
- However,-while-Kek5∆2$GFP- could-not- be- visualized-on- a-Western-blot- due- to- cell-
death- and- subsequent- protein- degredation,- the- cell- death- seen- upon- expression- of- this-
protein-is-a-highly-interesting-result.-The-stark-contrast-between-the-high-rates-of-cell-death-
seen- in- the-UASIKek5∆2IGFP- co$transfection-and- the-healthy- cells- seen- in- the-UASIKek5∆3I
GFP# co$transfection- provides- the- first- set- of- evidence- for- IC2- and- IC3- having- distinct-
functions- from- one- another.- Based- on- the- increased- cell- death- seen- in- cells- expressing-
Kek5∆2$GFP,-IC2-seems-to-be-more-crucial-to-Kek5-function,-and-the-previously-suggested-
notion-that-this-region-is-a-regulator-of-Kek5-itself-is-supported-by-the-data-presented-here.-
- The- generation- of- stable- stocks- of- pUASTaKek5∆2GFP# and# pUASTaKek5∆3GFP# is- a-
crucial-aspect-of-determining-IC2-and-IC3-function-in#vivo.-So-far,-F1-transgenics-have-been-
successfully- gathered- from- the- G0- crosses- set- up- with- w1118- from- the- original- injected-
larvae.-These-F1- transgenics,- signified-by- the-presence-of-eye- color,-have-been-crossed- to-
w1118- and- the- lab- is- still- awaiting- the- F2- generation- from- these- crosses.- Once- the- F2-
generation- is-produced,- the-chromosomal- location-of- the-Kek5∆2- or-Kek5∆3- gene- insertion-
27-
!
can-be-mapped-and-stable-stocks-can-be-generated.-These-stable-stocks-can-be-crossed-to-
A9.Gal4- or-Ptc.Gal4- flies- to- initiate-Kek5∆2- or-Kek5∆3- expression- in- the-offspring-of- such-a-
cross.- While- this- means- that- misexpression- studies- for- Kek5∆2- or- Kek5∆3- in- Drosophila#
melanogaster-are-still-a-few-months-off,-the-crucial-first-steps-in-this-process-have-already-
been-taken.-
In-past-studies,-cell-culture-has-not-been-used-as-a-tool-for-observing-misexpression-
effects- of- Kekkon5- and- its- many- deletion- variants.- This- study- has- shown- that- co$
transfection- of- UASIKek5IGFP# constructs- with- a- Gal4- driver- into- Drosophila# cells- can-
provide- a- relatively-quick- and- easy-method- for- assessing- some- functionalities- associated-
with-SLiMs-of-Kek5,-especially-compared-to-the-long-process-of-generating-stable-transgenic-
lines-in-Drosophila#melanogaster.-
- With-Kek5-playing-a-regulatory-role-in-BMP-signaling,-a-pathway-conserved-in-
vertebrates-that-influences-the-development-of-healthy-tissues-and-cancerous-cells-alike,-
the-characterization-of-Kek5-has-implications-for-combating-BMP$related-diseases-more-
effectively-and-better-understanding-vertebrate-development.-With-constructs-deleting-IC2-
and-IC3-of-Kek5-successfully-generated,-and-a-cell-death-activity-correlated-with-the-
deletion-of-the-SLiM-$-IC2,-a-key-link-between-the-intracellular-structure-and-sequence-of-
Kek5-to-it’s-function-has-been-elucidated.--With-the-extensive-array-of-intracellular-variants-
and-tools-to-assess-cell-death-pathways-readily-available,-rapid-progress-on-defining-the-
mechanism-underlying-the-role-of-IC2,-and-ultimately-Kek5,-in-cell-death/viability-can-now-
be-made.-
-
28-
!
REFERENCES,-
Berg-J.M.,-Tymoczko,-J.L.,-and-Stryer-L.-(2002).-Biochemistry.-New-York:-W.-H.-Freeman-and-Company.-
Bork,-P.,-Holm-L.-and-Sander,-C.-(1994).-The-Immunoglobulin-Fold:-Structural-Classification,-Sequence-Patterns-and-Common-Core.-J.#Mol.#Biol.#242,-309$320.-
Chen,-Y.,-Aulia,-S.,-Li,-L.-and-Tang,-B.L.-(2006).-AMIGO-And-Friends:-An-Emerging-Family-of-Brain$Enriched,-Neuronal-Growth-Modulating,-Type-I-Transmembrane-Proteins-with-Leucine$Rich-repeats-(LRR)-and-Cell-Adhesion-Molecule-Motifs.-Brain#Res.#Rev.#51,-265$274.-
Davey,-N.E.,-Sheilds,-D.C.,-and-Edwards,-R.J.-(2006).-SLiMDisc:-Short,-Linear-Motif-Discovery,-Correcting-for-Common-Evolutionary-Descent.-Nucleic#Acids#Res.#34,-3546$3554.--
Duffy,-J.B.-(2002).-GAL4-System-in-Drosophila:-A-Fly-Geneticist’s-Swiss-Army-Knife.-Genesis#34,-1$15.-
Evans,-T.-(2006).-Characterization-of-Kekkon5,-a-Drosophila#LIG-Protein-that-Modulates-BMP-and-Integrin-Function.-PhD-thesis.-Indiana-University.-
Evans,-T.,-Haridas,-H.,-and-Duffy,-J.B.-(2009).-Kekkon5-is-an-Extracellular-Regulator-of-BMP-Signaling.-Dev.#Biol.-326,-36$46.-
Klueg,-K.M.,-Alvarado,-D.,-Muskavitch,-M.A.T.,-Duffy,-J.B.-(2002).-Creation-of-a-GAL4/UAS$Coupled-Inducible-Gene-Expression-System-for-Use-in-Drosophila-Cultured-Cell-Lines.-Genesis#34,-119–122.-
Ko,-J.-and-Kim,-E.-(2007).-Leucine$Rich-Repeat-Proteins-of-Synapses.-J.#Neurosci.#Res.#85,-2824$2832.-
Little,-S.-and-Mullins,-M.-(2006).-Extracellular-Modulation-of-BMP-Activity-in-Patterning-the-Dorsoventral-Axis.-Birth#Defects#Res.,#Part#C#78,-224$242.-
MacLaren,-C.M.,-Evans,-T.A.,-Alvarado,-D.,-and-Duffy,-J.B.-(2004).-Comparative-Analysis-of-the-Kekkon-Molecules,-Related-Members-of-the-LIG-Superfamily.-Dev.#Genes#Evol.#214,-360$366.-
Mandai,-K.,-Guo,-T.,-St-Hillaire,-C.,-Meabon,-J.S.,-Kanning,-K.C.,-Bothwell,-M.,-and-Ginty,-D.D.-(2009).-LIG-Family-Receptor-Tyrosine-Kinase$Associated-Proteins-Modulate-Growth-Factor-Signals-during-Neural-Development.-Neuron#63,-614$627.-
Menon,-H.-(2013).-Deciphering-the-Role-of-Kekkon5-in-BMP-Signaling-and-Cell-Junction-Biology.-PhD-thesis.-Worcester-Polytechnic-Institute.--
29-
!
Musacchio,-M.,-and-Perrimon,-N.-(1996).-The-Drosophila-Kekkon-Genes:-Novel-Members-of-Both-the-Leucine$Rich-Repeat-and-Immunoglobulin-Superfamilies-Expressed-in-the-CNS.-Dev.#Biol.#178,-63$76.-
Ng,-A.C.,-Eisenberg,-J.M.,-Heath,-R.J.,-Huett,-A.,-Robinson,-C.M.,-Nau,-G.J.,-and-Xavier,-R.J.-(2010).-Human-Leucine$Rich-Repeat-Proteins:-A-Genome$Wide-Bioinformatic-Categorization-and-Functional-Analysis-in-Innate-Immunity.-Proc.#Natl.#Acad.#Sci.#USA#108,-Suppl.#1,-4631$4638.-
Padmanabhan,-M.,-Cournoyer,-P.,-and-Dinesh$Kumar,-S.P.-(2009).-The-Leucine$Rich-Repeat-Domain-in-Plant-Innate-Immunity:-A-Wealth-of-Possibilities.-Cell#Microbiol.#11,-191$198.-
-
30-
!
Appendix,A:,Site\Directed,Mutagenesis,Primer,Sequences,and,Locations,,,Primer#List#,
Primer,Name, Sequence, Description,W500- TAC-GGC-ATC-CGT-TCG-CCA-CCG-TCG-C- 5’-IC2-Flank-W501- TGG-CAA-GCT-ATT-GGC-ATA-CGC-ATC- 3’-IC2-Flank-W502- GAG-CCA-GTG-TAC-GAC-AAC-TTG-GGA-TTG-C- 5’-IC3-Flank-W503- CAC-ACC-CGT-GGC-CGG-CGA-ATA-GAC- 3’-IC3-Flank-
,Primer#Position#on#Kek5wt#
,,, ,
31-
!
Appendix,B:,Ongoing,Transgenic,F1,Crosses,in,Drosophila,,,
Kek5∆2,Transgenic,F1,Crosses, Kek5∆3,Transgenic,F1,Crosses,1A$2F$1M- 2A$11F$1F-1A$2F$2F- 2A$11F$2M-1A$4F$1M- 2A$16F$1F-1A$4F$2M- 2A$16F$2M-1A$4F$3M- 2A$16F$3M-1A$8M$1F- 2A$27F$1M-1A$8M$2M- 2A$27F$2F-1A$8M$3M- 2A$34F$1M-1A$9F$1F- 2A$34F$2F-1A$18M$1M- 2A$4F$1M-1A$18M$2M- 2A$18F$1F-1B$2F$1M- 2A$18F$2M-1B$2F$2M- 2A$22M$1M-1B$12M$1M- 2A$22M$2M-1B$12M$2F- 2B$4F$1M-- 2B$4F$2M-- 2B$6M$1F-- 2B$6M$2F-- 2C$25M$1M-, 2C$25M$2M-, 2C$29F$1M-,Notes:%All-lines-were-crossed-with-w1118-flies-of-the-opposite-sex.-The-first-section-of-each-transgenic-label-corresponds-to-the-vial-each-G0-originated-from.-The-second-section-of-each-label-corresponds-to-the-sex-of-the-parent-G0-and-the-order-in-which-it-eclosed-compared-to-its-siblings.-The-third-section-of-each-label-corresponds-to-the-sex-of-each-F1-transgenic-and-the-order-in-which-it-eclosed.--,,,,, ,
32-
!
Appendix,C:,GENE,CONSTRUCTION,CONSTRUCTS,,
CGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG
CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCT
TTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCTAGCCAGGAAGAGTTTGTAGAAACGCAAAAAGGCCA
TCCGTCAGGATGGCCTTCTGCTTAGTTTGATGCCTGGCAGTTTATGGCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTT
GCTTCACAACGTTCAAATCCGCTCCCGGCGGATTTGTCCTACTCAGGAGAGCGTTCACCGACAAACAACAGATAAAACG
AAAGGCCCAGTCTTCCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCAGTTCCCTACTCTCGCGTTAACGCTAGCATG
GATCTCGGGCCCCAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAAT
TGATGAGCAATGCTTTTTTATAATGCCAACTTTGTACAAAAAAGCAGGCTCCAGGAAA ATG
ATC CTT CTG CTG CTG GGT GTG CTA GTG GTT CTA ATG GCC CTA CCG CCG CCC ACC GCA GGC
ACC ACC GAT TGG ATG CAG AGC TGC GGT ACA TGC CAC TGT CAG TGG AAT TCG GGC AAG AAG
CTCGGAAAGCAAAATAAACTACGGACCGTCAAGGGATGAGAGCGCAATTGCGATCGTAC
CTAGAGCCCGGGGTTTATTACTAAAATAAAACTGACTATCACTGGACAAGCAACGTTGTTTA
ACTACTCGTTACGAAAAAATATTACGGTTGAAACATGTTTTTTCGTCCGAGGTCCTTT TAC
TAG GAA GAC GAC GAC CCA CAC GAT CAC CAA GAT TAC CGG GAT GGC GGC GGG TGG CGT CCG
TGG TGG CTA ACC TAC GTC TCG ACG CCA TGT ACG GTG ACA GTC ACC TTA AGC CCG TTC TTC
GCGGACCATAGAAATATCAGGACAGCCCAAAGCGGTGGAGACTGAACTCGCAGCTAAAAACACTACGAGCAGTCCCCCC
GCCTCGGATACCTTTTTGCGGTCGTTGCGCCGGAAAAATGCCAAGGACCGGAAAACGACCGGAAAACGAGTGTACAAGA
AAGGACGCAATAGGGGACTAAGACACCTATTGGCATAATGGCGATCGGTCCTTCTCAAACATCTTTGCGTTTTTCCGGT
AGGCAGTCCTACCGGAAGACGAATCAAACTACGGACCGTCAAATACCGCCCGCAGGACGGGCGGTGGGAGGCCCGGCAA
CGAAGTGTTGCAAGTTTAGGCGAGGGCCGCCTAAACAGGATGAGTCCTCTCGCAAGTGGCTGTTTGTTGTCTATTTTGC
TTTCCGGGTCAGAAGGCTGA
M
I L L L L G V L V V L M A L P P P T A G
T T D W M Q S C G T C H C Q W N S G K K
S A D C K N K A L T K I P Q D M S N E M
Q V L D F A H N Q I P E L R R E E F L L
A G L P N V H K I F L R N C T I Q E V H
R E A F K G L H I L I E L D L S G N R I
R E L H P G T F A G L E K L R N V I I N
N N E I E V L P N H L F V N L S F L S R
I E F R N N R L R Q V Q L H V F A G T M
A L S A I S L E Q N R L S H L H K E T F
K D L Q K L M H L S L Q G N A W N C S C
E L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N F
A C R P R I L G S V R S F I E A N H D N
I S L P C R I V G S P R P N V T W V Y N
K R P L Q Q Y D P R V R V L T S V E Q M
P E Q P S Q V L T S E L R I V G V R A S
D K G A Y T C V A D N R G G R A E A E F
Q L L V S G D Y A G A V S A S D G M G M
G A I G A P T I D P Q T N M F L I I C L
I I T T L L L L L L V A V L T L F W Y C
R R I K T Y Q K D T T M M S G D G L I S
S K M D K T H N G S M L E G S V I M E M
Q K S L L N E V N P V E K P P R R T D I
E S V D G G D D V L E I K K T L L D D T
V Y V A N H S R D E E A V S V A M S D T
T T T P R S R H T Y V D D A Y A N S L P
Y G I R S P P S L T S P V Y T H M T P H
G I Y G T K T M T A P H N G F M T L Q H
P K S R N L A L I A T T N S S R Q H Q H
H H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V V
Y S P A T G V V M K Q G Y M T I P R K P
R A P S W A P S T S G A A G H G S I Q L
S E F Q S P T S P N P S E T G T A T T A
E L Q A E P V Y D N L G L R T T A G G N
S T L N L T K I A G S Q G G A G Q Q Y S
M R D R P L P A T P S L T S V S S A T N
A S K I Y E P I H E L I Q Q Q Q Q L Q Q
Q Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G H
A A Y L S P G S G A A V S P S H A S S S
G D S P K A A K I P P R P P P K P K K K
M S V T T T R S G Q G S T S Q L F D D E
G E D G T E V
W50 Start of Good Sequence (B1, pENTRK5∆2)
NheI
NheI
attL1
junction markersignal sequence
KEK5
N FLANK
1
80
159
238
317
396
475
537
598
658
1
2
22
42
62
82
102
122
142
162
182
202
222
242
262
282
302
322
342
362
382
402
422
442
462
482
502
522
542
562
582
602
622
642
662
682
702
722
742
762
782
802
822
842
862
882
AGC GCC GAC TGC AAG AAC AAG GCG CTA ACC AAA ATT CCG CAG GAC ATG AGC AAC GAG ATG
CAG GTG CTG GAC TTT GCC CAC AAT CAA ATA CCC GAG CTG CGG CGC GAA GAG TTC CTA CTG
GCC GGT CTG CCC AAT GTG CAC AAG ATC TTT TTG CGC AAC TGC ACC ATC CAG GAG GTG CAT
CGC GAG GCC TTC AAG GGT CTG CAT ATC CTA ATC GAG CTG GAC CTG TCG GGC AAT CGG ATA
CGG GAA CTG CAT CCG GGC ACT TTC GCC GGC CTG GAG AAG CTG CGC AAC gTG ATC ATC AAC
AAC AAC GAG ATC GAG GTG CTG CCC AAC CAT CTG TTC GTC AAC CTG AGC TTC CTG TCG CGC
ATC GAG TTC CGG AAC AAT CGA TTG CGC CAG GTG CAG CTG CAC GTC TTC GCT GGC ACA ATG
GCG CTG AGC GCC ATT TCG CTG GAA CAG AAC CGC CTC TCA CAT CTG CAC AAG GAG ACA TTC
TCG CGG CTG ACG TTC TTG TTC CGC GAT TGG TTT TAA GGC GTC CTG TAC TCG TTG CTC TAC
GTC CAC GAC CTG AAA CGG GTG TTA GTT TAT GGG CTC GAC GCC GCG CTT CTC AAG GAT GAC
CGG CCA GAC GGG TTA CAC GTG TTC TAG AAA AAC GCG TTG ACG TGG TAG GTC CTC CAC GTA
GCG CTC CGG AAG TTC CCA GAC GTA TAG GAT TAG CTC GAC CTG GAC AGC CCG TTA GCC TAT
GCC CTT GAC GTA GGC CCG TGA AAG CGG CCG GAC CTC TTC GAC GCG TTG cAC TAG TAG TTG
TTG TTG CTC TAG CTC CAC GAC GGG TTG GTA GAC AAG CAG TTG GAC TCG AAG GAC AGC GCG
TAG CTC AAG GCC TTG TTA GCT AAC GCG GTC CAC GTC GAC GTG CAG AAG CGA CCG TGT TAC
CGC GAC TCG CGG TAA AGC GAC CTT GTC TTG GCG GAG AGT GTA GAC GTG TTC CTC TGT AAG
M
I L L L L G V L V V L M A L P P P T A G
T T D W M Q S C G T C H C Q W N S G K K
S A D C K N K A L T K I P Q D M S N E M
Q V L D F A H N Q I P E L R R E E F L L
A G L P N V H K I F L R N C T I Q E V H
R E A F K G L H I L I E L D L S G N R I
R E L H P G T F A G L E K L R N V I I N
N N E I E V L P N H L F V N L S F L S R
I E F R N N R L R Q V Q L H V F A G T M
A L S A I S L E Q N R L S H L H K E T F
K D L Q K L M H L S L Q G N A W N C S C
E L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N F
A C R P R I L G S V R S F I E A N H D N
I S L P C R I V G S P R P N V T W V Y N
K R P L Q Q Y D P R V R V L T S V E Q M
P E Q P S Q V L T S E L R I V G V R A S
D K G A Y T C V A D N R G G R A E A E F
Q L L V S G D Y A G A V S A S D G M G M
G A I G A P T I D P Q T N M F L I I C L
I I T T L L L L L L V A V L T L F W Y C
R R I K T Y Q K D T T M M S G D G L I S
S K M D K T H N G S M L E G S V I M E M
Q K S L L N E V N P V E K P P R R T D I
E S V D G G D D V L E I K K T L L D D T
V Y V A N H S R D E E A V S V A M S D T
T T T P R S R H T Y V D D A Y A N S L P
Y G I R S P P S L T S P V Y T H M T P H
G I Y G T K T M T A P H N G F M T L Q H
P K S R N L A L I A T T N S S R Q H Q H
H H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V V
Y S P A T G V V M K Q G Y M T I P R K P
R A P S W A P S T S G A A G H G S I Q L
S E F Q S P T S P N P S E T G T A T T A
E L Q A E P V Y D N L G L R T T A G G N
S T L N L T K I A G S Q G G A G Q Q Y S
M R D R P L P A T P S L T S V S S A T N
A S K I Y E P I H E L I Q Q Q Q Q L Q Q
Q Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G H
A A Y L S P G S G A A V S P S H A S S S
G D S P K A A K I P P R P P P K P K K K
M S V T T T R S G Q G S T S Q L F D D E
G E D G T E V
LRRs
BglII
Actual sequence is GTG - should match original wt sequence
oligo238
718
778
838
898
958
1018
1078
1138
1
2
22
42
62
82
102
122
142
162
182
202
222
242
262
282
302
322
342
362
382
402
422
442
462
482
502
522
542
562
582
602
622
642
662
682
702
722
742
762
782
802
822
842
862
882
AAG GAT CTG CAG AAG CTG ATG CAT CTA TCG CTG CAG GGT AAC GCA TGG AAC TGC AGC TGC
GAG CTG CAG GAC TTT CGC GAC TTT GCG ATC AGC AAA CGG CTC TAC ACA CCG CCC ACC GAT
TGC CAG GAG CCG CCA CAG CTG CGC GGC AAG CTG TGG AGC GAG GTG CCA TCG GAG AAC TTC
GCC TGC CGG CCG CGC ATT TTG GGT TCC GTG CGC TCC TTC ATC GAG GCC AAT CAC GAC AAT
ATC TCG CTA CCC TGC CGC ATT GTC GGC AGT CCG CGT CCC AAT GTC ACC TGG GTG TAC AAC
AAG CGG CCA TTG CAG CAG TAC GAC CCG CGT GTG CGT GTC CTC ACC TCC GTG GAA CAG ATG
CCG GAG CAG CCC TCC CAG GTG CTC ACC TCG GAG CTG CGC ATC GTG GGC GTA CGG GCC TCC
GAC AAG GGT GCC TAC ACC TGT GTG GCG GAT AAC CGG GGC GGA CGG GCG GAG GCC GAG TTC
TTC CTA GAC GTC TTC GAC TAC GTA GAT AGC GAC GTC CCA TTG CGT ACC TTG ACG TCG ACG
CTC GAC GTC CTG AAA GCG CTG AAA CGC TAG TCG TTT GCC GAG ATG TGT GGC GGG TGG CTA
ACG GTC CTC GGC GGT GTC GAC GCG CCG TTC GAC ACC TCG CTC CAC GGT AGC CTC TTG AAG
CGG ACG GCC GGC GCG TAA AAC CCA AGG CAC GCG AGG AAG TAG CTC CGG TTA GTG CTG TTA
TAG AGC GAT GGG ACG GCG TAA CAG CCG TCA GGC GCA GGG TTA CAG TGG ACC CAC ATG TTG
TTC GCC GGT AAC GTC GTC ATG CTG GGC GCA CAC GCA CAG GAG TGG AGG CAC CTT GTC TAC
GGC CTC GTC GGG AGG GTC CAC GAG TGG AGC CTC GAC GCG TAG CAC CCG CAT GCC CGG AGG
CTG TTC CCA CGG ATG TGG ACA CAC CGC CTA TTG GCC CCG CCT GCC CGC CTC CGG CTC AAG
M
I L L L L G V L V V L M A L P P P T A G
T T D W M Q S C G T C H C Q W N S G K K
S A D C K N K A L T K I P Q D M S N E M
Q V L D F A H N Q I P E L R R E E F L L
A G L P N V H K I F L R N C T I Q E V H
R E A F K G L H I L I E L D L S G N R I
R E L H P G T F A G L E K L R N V I I N
N N E I E V L P N H L F V N L S F L S R
I E F R N N R L R Q V Q L H V F A G T M
A L S A I S L E Q N R L S H L H K E T F
K D L Q K L M H L S L Q G N A W N C S C
E L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N F
A C R P R I L G S V R S F I E A N H D N
I S L P C R I V G S P R P N V T W V Y N
K R P L Q Q Y D P R V R V L T S V E Q M
P E Q P S Q V L T S E L R I V G V R A S
D K G A Y T C V A D N R G G R A E A E F
Q L L V S G D Y A G A V S A S D G M G M
G A I G A P T I D P Q T N M F L I I C L
I I T T L L L L L L V A V L T L F W Y C
R R I K T Y Q K D T T M M S G D G L I S
S K M D K T H N G S M L E G S V I M E M
Q K S L L N E V N P V E K P P R R T D I
E S V D G G D D V L E I K K T L L D D T
V Y V A N H S R D E E A V S V A M S D T
T T T P R S R H T Y V D D A Y A N S L P
Y G I R S P P S L T S P V Y T H M T P H
G I Y G T K T M T A P H N G F M T L Q H
P K S R N L A L I A T T N S S R Q H Q H
H H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V V
Y S P A T G V V M K Q G Y M T I P R K P
R A P S W A P S T S G A A G H G S I Q L
S E F Q S P T S P N P S E T G T A T T A
E L Q A E P V Y D N L G L R T T A G G N
S T L N L T K I A G S Q G G A G Q Q Y S
M R D R P L P A T P S L T S V S S A T N
A S K I Y E P I H E L I Q Q Q Q Q L Q Q
Q Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G H
A A Y L S P G S G A A V S P S H A S S S
G D S P K A A K I P P R P P P K P K K K
M S V T T T R S G Q G S T S Q L F D D E
G E D G T E V
oligo594
C FLANK
Ig
1198
1258
1318
1378
1438
1498
1558
1618
1
2
22
42
62
82
102
122
142
162
182
202
222
242
262
282
302
322
342
362
382
402
422
442
462
482
502
522
542
562
582
602
622
642
662
682
702
722
742
762
782
802
822
842
862
882
CAG CTG CTC GTG AGC GGT GAC TAT GCC GGC GCG GTA TCC GCC TCC GAT GGC ATG GGC ATG
GGC GCC ATT GGG GCA CCA ACC ATT GAT CCG CAA ACG AAC ATG TTT CTC ATC ATC TGT CTA
ATC ATT ACG ACG CTG CTG CTC CTG CTG CTC GTG GCG GTG CTG ACG CTC TTC TGG TAC TGC
CGT CGC ATC AAG ACC TAT CAA AAG GAC ACC ACC ATG ATG AGC GGC GAC GGG CTG ATC TCT
TCC AAG ATG GAC AAG ACG CAC AAC GGC TCC ATG CTC GAG GGT TCC GTC ATC ATG GAG ATG
CAG AAG AGC CTG CTC AAC GAG GTC AAT CCA GTC GAG AAG CCG CCA CGG CGC ACG GAC ATC
GTC GAC GAG CAC TCG CCA CTG ATA CGG CCG CGC CAT AGG CGG AGG CTA CCG TAC CCG TAC
CCG CGG TAA CCC CGT GGT TGG TAA CTA GGC GTT TGC TTG TAC AAA GAG TAG TAG ACA GAT
TAG TAA TGC TGC GAC GAC GAG GAC GAC GAG CAC CGC CAC GAC TGC GAG AAG ACC ATG ACG
GCA GCG TAG TTC TGG ATA GTT TTC CTG TGG TGG TAC TAC TCG CCG CTG CCC GAC TAG AGA
AGG TTC TAC CTG TTC TGC GTG TTG CCG AGG TAC GAG CTC CCA AGG CAG TAG TAC CTC TAC
GTC TTC TCG GAC GAG TTG CTC CAG TTA GGT CAG CTC TTC GGC GGT GCC GCG TGC CTG TAG
M
I L L L L G V L V V L M A L P P P T A G
T T D W M Q S C G T C H C Q W N S G K K
S A D C K N K A L T K I P Q D M S N E M
Q V L D F A H N Q I P E L R R E E F L L
A G L P N V H K I F L R N C T I Q E V H
R E A F K G L H I L I E L D L S G N R I
R E L H P G T F A G L E K L R N V I I N
N N E I E V L P N H L F V N L S F L S R
I E F R N N R L R Q V Q L H V F A G T M
A L S A I S L E Q N R L S H L H K E T F
K D L Q K L M H L S L Q G N A W N C S C
E L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N F
A C R P R I L G S V R S F I E A N H D N
I S L P C R I V G S P R P N V T W V Y N
K R P L Q Q Y D P R V R V L T S V E Q M
P E Q P S Q V L T S E L R I V G V R A S
D K G A Y T C V A D N R G G R A E A E F
Q L L V S G D Y A G A V S A S D G M G M
G A I G A P T I D P Q T N M F L I I C L
I I T T L L L L L L V A V L T L F W Y C
R R I K T Y Q K D T T M M S G D G L I S
S K M D K T H N G S M L E G S V I M E M
Q K S L L N E V N P V E K P P R R T D I
E S V D G G D D V L E I K K T L L D D T
V Y V A N H S R D E E A V S V A M S D T
T T T P R S R H T Y V D D A Y A N S L P
Y G I R S P P S L T S P V Y T H M T P H
G I Y G T K T M T A P H N G F M T L Q H
P K S R N L A L I A T T N S S R Q H Q H
H H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V V
Y S P A T G V V M K Q G Y M T I P R K P
R A P S W A P S T S G A A G H G S I Q L
S E F Q S P T S P N P S E T G T A T T A
E L Q A E P V Y D N L G L R T T A G G N
S T L N L T K I A G S Q G G A G Q Q Y S
M R D R P L P A T P S L T S V S S A T N
A S K I Y E P I H E L I Q Q Q Q Q L Q Q
Q Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G H
A A Y L S P G S G A A V S P S H A S S S
G D S P K A A K I P P R P P P K P K K K
M S V T T T R S G Q G S T S Q L F D D E
G E D G T E V
end Ig?
Tm
IC
∆123 Confirmed∆1235 Confirmed∆1234 Confirmed
oligo745 apK5_seq_int_5'
IC1 modified
oligo746 apK5_seq_int_3'
∆1 ConfirmedIC1
1678
1738
1798
1858
1918
1978
1
2
22
42
62
82
102
122
142
162
182
202
222
242
262
282
302
322
342
362
382
402
422
442
462
482
502
522
542
562
582
602
622
642
662
682
702
722
742
762
782
802
822
842
862
882
GAG AGC GTG GAT GGT GGC GAT GAC GTG CTC GAG ATC AAG AAG ACG CTG CTC GAC GAC ACC
GTC TAT GTG GCC AAT CAC TCG CGC GAC GAA GAA GCC GTC TCA GTG GCC ATG TCG GAT ACG
ACG ACC ACG CCC CGA TCT CGA CAC ACC TAC GTG GAT GAT GCG TAT GCC AAT AGC TTG CCA
TAC GGC ATC CGT TCG CCA CCG TCG CTA ACC AGT CCG GTC TAC ACG CAT ATG ACG CCG CAC
GGC ATC TAC GGC ACC AAG ACG ATG ACG GCT CCG CAT AAC GGC TTT ATG ACG CTG CAG CAT
CCC AAG TCG CGC AAC CTG GCG CTC ATT GCC ACC ACC AAC AGC AGT CGC CAG CAC CAG CAC
CAC CAT CAG CTG CAG CAG CAG CAG CAG CAC CAC CAC CAC CAC CAG CAG CAA CAA CAA CAG
CAG CAG CAG CAG CAA CAT CCG CTG GCC ACC ACA TCG CCC TTC CTG CCC GCA CCC GTC GTC
CTC TCG CAC CTA CCA CCG CTA CTG CAC GAG CTC TAG TTC TTC TGC GAC GAG CTG CTG TGG
CAG ATA CAC CGG TTA GTG AGC GCG CTG CTT CTT CGG CAG AGT CAC CGG TAC AGC CTA TGC
TGC TGG TGC GGG GCT AGA GCT GTG TGG ATG CAC CTA CTA CGC ATA CGG TTA TCG AAC GGT
ATG CCG TAG GCA AGC GGT GGC AGC GAT TGG TCA GGC CAG ATG TGC GTA TAC TGC GGC GTG
CCG TAG ATG CCG TGG TTC TGC TAC TGC CGA GGC GTA TTG CCG AAA TAC TGC GAC GTC GTA
GGG TTC AGC GCG TTG GAC CGC GAG TAA CGG TGG TGG TTG TCG TCA GCG GTC GTG GTC GTG
GTG GTA GTC GAC GTC GTC GTC GTC GTC GTG GTG GTG GTG GTG GTC GTC GTT GTT GTT GTC
GTC GTC GTC GTC GTT GTA GGC GAC CGG TGG TGT AGC GGG AAG GAC GGG CGT GGG CAG CAG
M
I L L L L G V L V V L M A L P P P T A G
T T D W M Q S C G T C H C Q W N S G K K
S A D C K N K A L T K I P Q D M S N E M
Q V L D F A H N Q I P E L R R E E F L L
A G L P N V H K I F L R N C T I Q E V H
R E A F K G L H I L I E L D L S G N R I
R E L H P G T F A G L E K L R N V I I N
N N E I E V L P N H L F V N L S F L S R
I E F R N N R L R Q V Q L H V F A G T M
A L S A I S L E Q N R L S H L H K E T F
K D L Q K L M H L S L Q G N A W N C S C
E L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N F
A C R P R I L G S V R S F I E A N H D N
I S L P C R I V G S P R P N V T W V Y N
K R P L Q Q Y D P R V R V L T S V E Q M
P E Q P S Q V L T S E L R I V G V R A S
D K G A Y T C V A D N R G G R A E A E F
Q L L V S G D Y A G A V S A S D G M G M
G A I G A P T I D P Q T N M F L I I C L
I I T T L L L L L L V A V L T L F W Y C
R R I K T Y Q K D T T M M S G D G L I S
S K M D K T H N G S M L E G S V I M E M
Q K S L L N E V N P V E K P P R R T D I
E S V D G G D D V L E I K K T L L D D T
V Y V A N H S R D E E A V S V A M S D T
T T T P R S R H T Y V D D A Y A N S L P
Y G I R S P P S L T S P V Y T H M T P H
G I Y G T K T M T A P H N G F M T L Q H
P K S R N L A L I A T T N S S R Q H Q H
H H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V V
Y S P A T G V V M K Q G Y M T I P R K P
R A P S W A P S T S G A A G H G S I Q L
S E F Q S P T S P N P S E T G T A T T A
E L Q A E P V Y D N L G L R T T A G G N
S T L N L T K I A G S Q G G A G Q Q Y S
M R D R P L P A T P S L T S V S S A T N
A S K I Y E P I H E L I Q Q Q Q Q L Q Q
Q Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G H
A A Y L S P G S G A A V S P S H A S S S
G D S P K A A K I P P R P P P K P K K K
M S V T T T R S G Q G S T S Q L F D D E
G E D G T E V
oligo240
∆IC2 ChloeL
2038
2098
2158
2218
2278
2338
2398
2458
1
2
22
42
62
82
102
122
142
162
182
202
222
242
262
282
302
322
342
362
382
402
422
442
462
482
502
522
542
562
582
602
622
642
662
682
702
722
742
762
782
802
822
842
862
882
TAT TCG CCG GCC ACG GGT GTG GTC ATG AAA CAG GGA TAT ATG ACC ATT CCG CGC AAG CCG
CGC GCT CCC AGC TGG GCG CCC AGT ACT TCC GGT GCC GCT GGC CAC GGA TCC ATT CAG CTA
AGT GAA TTC CAG AGC CCC ACA TCG CCG AAT CCC AGC GAG ACT GGC ACC GCC ACC ACC GCG
GAA CTG CAG GCG GAG CCA GTG TAC GAC AAC TTG GGA TTG CGA ACC ACT GCC GGC GGC AAC
TCC ACC CTC AAT CTG ACC AAG ATC GCC GGC TCA CAG GGG GGC GCT GGT CAG CAG TAC TCG
ATG CGG GAC CGA CCA CTT CCG GCC ACG CCC AGC CTG ACA TCG GTG TCC TCG GCG ACC AAT
GCC AGT AAG ATT TAC GAG CCC ATA CAC GAG CTG ATT CAG CAG CAA CAG CAG TTG CAA CAA
ATA AGC GGC CGG TGC CCA CAC CAG TAC TTT GTC CCT ATA TAC TGG TAA GGC GCG TTC GGC
GCG CGA GGG TCG ACC CGC GGG TCA TGA AGG CCA CGG CGA CCG GTG CCT AGG TAA GTC GAT
TCA CTT AAG GTC TCG GGG TGT AGC GGC TTA GGG TCG CTC TGA CCG TGG CGG TGG TGG CGC
CTT GAC GTC CGC CTC GGT CAC ATG CTG TTG AAC CCT AAC GCT TGG TGA CGG CCG CCG TTG
AGG TGG GAG TTA GAC TGG TTC TAG CGG CCG AGT GTC CCC CCG CGA CCA GTC GTC ATG AGC
TAC GCC CTG GCT GGT GAA GGC CGG TGC GGG TCG GAC TGT AGC CAC AGG AGC CGC TGG TTA
CGG TCA TTC TAA ATG CTC GGG TAT GTG CTC GAC TAA GTC GTC GTT GTC GTC AAC GTT GTT
M
I L L L L G V L V V L M A L P P P T A G
T T D W M Q S C G T C H C Q W N S G K K
S A D C K N K A L T K I P Q D M S N E M
Q V L D F A H N Q I P E L R R E E F L L
A G L P N V H K I F L R N C T I Q E V H
R E A F K G L H I L I E L D L S G N R I
R E L H P G T F A G L E K L R N V I I N
N N E I E V L P N H L F V N L S F L S R
I E F R N N R L R Q V Q L H V F A G T M
A L S A I S L E Q N R L S H L H K E T F
K D L Q K L M H L S L Q G N A W N C S C
E L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N F
A C R P R I L G S V R S F I E A N H D N
I S L P C R I V G S P R P N V T W V Y N
K R P L Q Q Y D P R V R V L T S V E Q M
P E Q P S Q V L T S E L R I V G V R A S
D K G A Y T C V A D N R G G R A E A E F
Q L L V S G D Y A G A V S A S D G M G M
G A I G A P T I D P Q T N M F L I I C L
I I T T L L L L L L V A V L T L F W Y C
R R I K T Y Q K D T T M M S G D G L I S
S K M D K T H N G S M L E G S V I M E M
Q K S L L N E V N P V E K P P R R T D I
E S V D G G D D V L E I K K T L L D D T
V Y V A N H S R D E E A V S V A M S D T
T T T P R S R H T Y V D D A Y A N S L P
Y G I R S P P S L T S P V Y T H M T P H
G I Y G T K T M T A P H N G F M T L Q H
P K S R N L A L I A T T N S S R Q H Q H
H H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V V
Y S P A T G V V M K Q G Y M T I P R K P
R A P S W A P S T S G A A G H G S I Q L
S E F Q S P T S P N P S E T G T A T T A
E L Q A E P V Y D N L G L R T T A G G N
S T L N L T K I A G S Q G G A G Q Q Y S
M R D R P L P A T P S L T S V S S A T N
A S K I Y E P I H E L I Q Q Q Q Q L Q Q
Q Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G H
A A Y L S P G S G A A V S P S H A S S S
G D S P K A A K I P P R P P P K P K K K
M S V T T T R S G Q G S T S Q L F D D E
G E D G T E V
IC3 modified IC3
oligo241
∆45 Confirmed∆4 ConfirmedIC4 modified
IC4
2518
2578
2638
2698
2758
2818
2878
1
2
22
42
62
82
102
122
142
162
182
202
222
242
262
282
302
322
342
362
382
402
422
442
462
482
502
522
542
562
582
602
622
642
662
682
702
722
742
762
782
802
822
842
862
882
CAA CAA CAG CAG CAG CAG CAG CGA CTG GGC TCC ATG GAC ACG GAA CCC CTG TAC GGA GTT
CGG CAA CAG GGG ATC ACG ATA CTG CCC GGC TCG AGC ATT AGC GGT GCC GGA CTG GGC CAC
GCC GCC TAC CTT TCA CCC GGC TCG GGT GCC GCC GTA TCG CCA AGC CAC GCC AGC AGC AGC
GGT GAC TCT CCG AAG GCC GCC AAG ATC CCA CCA CGC CCA CCA CCG AAG CCC AAG AAG AAG
ATG TCC GTG ACG ACG ACG CGC AGC GGC CAG GGC AGC ACC AGC CAG CTC TTC GAC GAC GAG
GGC GAG GAT GGC ACC GAG GTC G ACCCAGCTTTCTTGTACAAAGTTGGCATTATAAGAA
AGCATTGCTTATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATT
ATTTGCCATCCAGCTGCAGCTCTGGCCCGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCAT
GTT GTT GTC GTC GTC GTC GTC GCT GAC CCG AGG TAC CTG TGC CTT GGG GAC ATG CCT CAA
GCC GTT GTC CCC TAG TGC TAT GAC GGG CCG AGC TCG TAA TCG CCA CGG CCT GAC CCG GTG
CGG CGG ATG GAA AGT GGG CCG AGC CCA CGG CGG CAT AGC GGT TCG GTG CGG TCG TCG TCG
CCA CTG AGA GGC TTC CGG CGG TTC TAG GGT GGT GCG GGT GGT GGC TTC GGG TTC TTC TTC
TAC AGG CAC TGC TGC TGC GCG TCG CCG GTC CCG TCG TGG TCG GTC GAG AAG CTG CTG CTC
CCG CTC CTA CCG TGG CTC CAG C TGGGTCGAAAGAACATGTTTCAACCGTAATATTCTT
TCGTAACGAATAGTTAAACAACGTTGCTTGTCCAGTGATAGTCAGTTTTATTTTAGTAA
TAAACGGTAGGTCGACGTCGAGACCGGGCACAGAGTTTTAGAGACTACAATGTAACGTGTTCTATTTTTATATAGTA
M
I L L L L G V L V V L M A L P P P T A G
T T D W M Q S C G T C H C Q W N S G K K
S A D C K N K A L T K I P Q D M S N E M
Q V L D F A H N Q I P E L R R E E F L L
A G L P N V H K I F L R N C T I Q E V H
R E A F K G L H I L I E L D L S G N R I
R E L H P G T F A G L E K L R N V I I N
N N E I E V L P N H L F V N L S F L S R
I E F R N N R L R Q V Q L H V F A G T M
A L S A I S L E Q N R L S H L H K E T F
K D L Q K L M H L S L Q G N A W N C S C
E L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N F
A C R P R I L G S V R S F I E A N H D N
I S L P C R I V G S P R P N V T W V Y N
K R P L Q Q Y D P R V R V L T S V E Q M
P E Q P S Q V L T S E L R I V G V R A S
D K G A Y T C V A D N R G G R A E A E F
Q L L V S G D Y A G A V S A S D G M G M
G A I G A P T I D P Q T N M F L I I C L
I I T T L L L L L L V A V L T L F W Y C
R R I K T Y Q K D T T M M S G D G L I S
S K M D K T H N G S M L E G S V I M E M
Q K S L L N E V N P V E K P P R R T D I
E S V D G G D D V L E I K K T L L D D T
V Y V A N H S R D E E A V S V A M S D T
T T T P R S R H T Y V D D A Y A N S L P
Y G I R S P P S L T S P V Y T H M T P H
G I Y G T K T M T A P H N G F M T L Q H
P K S R N L A L I A T T N S S R Q H Q H
H H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V V
Y S P A T G V V M K Q G Y M T I P R K P
R A P S W A P S T S G A A G H G S I Q L
S E F Q S P T S P N P S E T G T A T T A
E L Q A E P V Y D N L G L R T T A G G N
S T L N L T K I A G S Q G G A G Q Q Y S
M R D R P L P A T P S L T S V S S A T N
A S K I Y E P I H E L I Q Q Q Q Q L Q Q
Q Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G H
A A Y L S P G S G A A V S P S H A S S S
G D S P K A A K I P P R P P P K P K K K
M S V T T T R S G Q G S T S Q L F D D E
G E D G T E V
∆1235 Confirmed∆5 ConfirmedIC5
IC6 (PDZ)
attB2attL2
End of Good Sequence (B1, pENTRK5∆2) OLIGO 691
2938
2998
3058
3118
3178
3238
3296
3355
1
2
22
42
62
82
102
122
142
162
182
202
222
242
262
282
302
322
342
362
382
402
422
442
462
482
502
522
542
562
582
602
622
642
662
682
702
722
742
762
782
802
822
842
862
882
CATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCGAG
GCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCG
ACAATCTATCGCTTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATG
TTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCC
TGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGT
GAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCG
ATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCG
TAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCAT
GGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCG
CAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCA
AAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTG
GTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGCAAGCTCATGACCAAAATCCCTTAACG
TGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTA
ATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTC
CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAA
GAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGT
CTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC
CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGG
GAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAA
GTACTTGTTATTTTGACAGACGAATGTATTTGTCATTATGTTCCCCACAATACTCGGTATAAGTTGCCCTTTGCAGCTC
CGGCGCTAATTTAAGGTTGTACCTACGACTAAATATACCCATATTTACCCGAGCGCTATTACAGCCCGTTAGTCCACGC
TGTTAGATAGCGAACATACCCTTCGGGCTACGCGGTCTCAACAAAGACTTTGTACCGTTTCCATCGCAACGGTTACTAC
AATGTCTACTCTACCAGTCTGATTTGACCGACTGCCTTAAATACGGAGAAGGCTGGTAGTTCGTAAAATAGGCATGAGG
ACTACTACGTACCAATGAGTGGTGACGCTAGGGGCCTTTTTGTCGTAAGGTCCATAATCTTCTTATAGGACTAAGTCCA
CTTTTATAACAACTACGCGACCGTCACAAGGACGCGGCCAACGTAAGCTAAGGACAAACATTAACAGGAAAATTGTCGC
TAGCGCATAAAGCAGAGCGAGTCCGCGTTAGTGCTTACTTATTGCCAAACCAACTACGCTCACTAAAACTACTGCTCGC
ATTACCGACCGGACAACTTGTTCAGACCTTTCTTTACGTATTTGAAAACGGTAAGAGTGGCCTAAGTCAGCAGTGAGTA
CCACTAAAGAGTGAACTATTGGAATAAAAACTGCTCCCCTTTAATTATCCAACATAACTACAACCTGCTCAGCCTTAGC
GTCTGGCTATGGTCCTAGAACGGTAGGATACCTTGACGGAGCCACTCAAAAGAGGAAGTAATGTCTTTGCCGAAAAAGT
TTTTATACCATAACTATTAGGACTATACTTATTTAACGTCAAAGTAAACTACGAGCTACTCAAAAAGATTAGTCTTAAC
CAATTAACCAACATTGTGACCGTCTCGTAATGCGACTGAACTGCCCTGCCGCGTTCGAGTACTGGTTTTAGGGAATTGC
ACTCAAAAGCAAGGTGACTCGCAGTCTGGGGCATCTTTTCTAGTTTCCTAGAAGAACTCTAGGAAAAAAAGACGCGCAT
TAGACGACGAACGTTTGTTTTTTTGGTGGCGATGGTCGCCACCAAACAAACGGCCTAGTTCTCGATGGTTGAGAAAAAG
GCTTCCATTGACCGAAGTCGTCTCGCGTCTATGGTTTATGACAGGAAGATCACATCGGCATCAATCCGGTGGTGAAGTT
CTTGAGACATCGTGGCGGATGTATGGAGCGAGACGATTAGGACAATGGTCACCGACGACGGTCACCGCTATTCAGCACA
GAATGGCCCAACCTGAGTTCTGCTATCAATGGCCTATTCCGCGTCGCCAGCCCGACTTGCCCCCCAAGCACGTGTGTCG
GGTCGAACCTCGCTTGCTGGATGTGGCTTGACTCTATGGATGTCGCACTCGATACTCTTTCGCGGTGCGAAGGGCTTCC
CTCTTTCCGCCTGTCCATAGGCCATTCGCCGTCCCAGCCTTGTCCTCTCGCGTGCTCCCTCGAAGGTCCCCCTTT
3432
3511
3590
3669
3748
3827
3906
3985
4064
4143
4222
4301
4380
4459
4538
4617
4696
4775
4854
CCA TCC AGC TGC AGC TCT GGC CCG TGT CTC AAA ATC TCT GAT GTT ACA TTG CAC AAG ATA
AAA ATA TAT CAT CAT GAA CAA TAA AAC TGT CTG CTT ACA TAA ACA GTA ATA CAA GGG GTG
TTA TGA GCC ATA TTC AAC GGG AAA CGT CGA GGC CGC GAT TAA ATT CCA ACA TGG ATG CTG
ATT TAT ATG GGT ATA AAT GGG CTC GCG ATA ATG TCG GGC AAT CAG GTG CGA CAA TCT ATC
GCT TGT ATG GGA AGC CCG ATG CGC CAG AGT TGT TTC TGA AAC ATG GCA AAG GTA GCG TTG
CCA ATG ATG TTA CAG ATG AGA TGG TCA GAC TAA ACT GGC TGA CGG AAT TTA TGC CTC TTC
CGA CCA TCA AGC ATT TTA TCC GTA CTC CTG ATG ATG CAT GGT TAC TCA CCA CTG CGA TCC
CCG GAA AAA CAG CAT TCC AGG TAT TAG AAG AAT ATC CTG ATT CAG GTG AAA ATA TTG TTG
ATG CGC TGG CAG TGT TCC TGC GCC GGT TGC ATT CGA TTC CTG TTT GTA ATT GTC CTT TTA
ACA GCG ATC GCG TAT TTC GTC TCG CTC AGG CGC AAT CAC GAA TGA ATA ACG GTT TGG TTG
ATG CGA GTG ATT TTG ATG ACG AGC GTA ATG GCT GGC CTG TTG AAC AAG TCT GGA AAG AAA
TGC ATA AAC TTT TGC CAT TCT CAC CGG ATT CAG TCG TCA CTC ATG GTG ATT TCT CAC TTG
ATA ACC TTA TTT TTG ACG AGG GGA AAT TAA TAG GTT GTA TTG ATG TTG GAC GAG TCG GAA
TCG CAG ACC GAT ACC AGG ATC TTG CCA TCC TAT GGA ACT GCC TCG GTG AGT TTT CTC CTT
CAT TAC AGA AAC GGC TTT TTC AAA AAT ATG GTA TTG ATA ATC CTG ATA TGA ATA AAT TGC
AGT TTC ATT TGA TGC TCG ATG AGT TTT TCT AAT CAG AAT TGG TTA ATT GGT TGT AAC ACT
GGC AGA GCA TTA CGC TGA CTT GAC GGG ACG GCG CAA GCT CAT GAC CAA AAT CCC TTA ACG
TGA GTT TTC GTT CCA CTG AGC GTC AGA CCC CGT AGA AAA GAT CAA AGG ATC TTC TTG AGA
TCC TTT TTT TCT GCG CGT AAT CTG CTG CTT GCA AAC AAA AAA ACC ACC GCT ACC AGC GGT
GGT TTG TTT GCC GGA TCA AGA GCT ACC AAC TCT TTT TCC GAA GGT AAC TGG CTT CAG CAG
AGC GCA GAT ACC AAA TAC TGT CCT TCT AGT GTA GCC GTA GTT AGG CCA CCA CTT CAA GAA
GGT AGG TCG ACG TCG AGA CCG GGC ACA GAG TTT TAG AGA CTA CAA TGT AAC GTG TTC TAT
TTT TAT ATA GTA GTA CTT GTT ATT TTG ACA GAC GAA TGT ATT TGT CAT TAT GTT CCC CAC
AAT ACT CGG TAT AAG TTG CCC TTT GCA GCT CCG GCG CTA ATT TAA GGT TGT ACC TAC GAC
TAA ATA TAC CCA TAT TTA CCC GAG CGC TAT TAC AGC CCG TTA GTC CAC GCT GTT AGA TAG
CGA ACA TAC CCT TCG GGC TAC GCG GTC TCA ACA AAG ACT TTG TAC CGT TTC CAT CGC AAC
GGT TAC TAC AAT GTC TAC TCT ACC AGT CTG ATT TGA CCG ACT GCC TTA AAT ACG GAG AAG
GCT GGT AGT TCG TAA AAT AGG CAT GAG GAC TAC TAC GTA CCA ATG AGT GGT GAC GCT AGG
GGC CTT TTT GTC GTA AGG TCC ATA ATC TTC TTA TAG GAC TAA GTC CAC TTT TAT AAC AAC
TAC GCG ACC GTC ACA AGG ACG CGG CCA ACG TAA GCT AAG GAC AAA CAT TAA CAG GAA AAT
TGT CGC TAG CGC ATA AAG CAG AGC GAG TCC GCG TTA GTG CTT ACT TAT TGC CAA ACC AAC
TAC GCT CAC TAA AAC TAC TGC TCG CAT TAC CGA CCG GAC AAC TTG TTC AGA CCT TTC TTT
ACG TAT TTG AAA ACG GTA AGA GTG GCC TAA GTC AGC AGT GAG TAC CAC TAA AGA GTG AAC
TAT TGG AAT AAA AAC TGC TCC CCT TTA ATT ATC CAA CAT AAC TAC AAC CTG CTC AGC CTT
AGC GTC TGG CTA TGG TCC TAG AAC GGT AGG ATA CCT TGA CGG AGC CAC TCA AAA GAG GAA
GTA ATG TCT TTG CCG AAA AAG TTT TTA TAC CAT AAC TAT TAG GAC TAT ACT TAT TTA ACG
TCA AAG TAA ACT ACG AGC TAC TCA AAA AGA TTA GTC TTA ACC AAT TAA CCA ACA TTG TGA
CCG TCT CGT AAT GCG ACT GAA CTG CCC TGC CGC GTT CGA GTA CTG GTT TTA GGG AAT TGC
ACT CAA AAG CAA GGT GAC TCG CAG TCT GGG GCA TCT TTT CTA GTT TCC TAG AAG AAC TCT
AGG AAA AAA AGA CGC GCA TTA GAC GAC GAA CGT TTG TTT TTT TGG TGG CGA TGG TCG CCA
CCA AAC AAA CGG CCT AGT TCT CGA TGG TTG AGA AAA AGG CTT CCA TTG ACC GAA GTC GTC
TCG CGT CTA TGG TTT ATG ACA GGA AGA TCA CAT CGG CAT CAA TCC GGT GGT GAA GTT CTT
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
CTC TGT AGC ACC GCC TAC ATA CCT CGC TCT GCT AAT CCT GTT ACC AGT GGC TGC TGC CAG
TGG CGA TAA GTC GTG TCT TAC CGG GTT GGA CTC AAG ACG ATA GTT ACC GGA TAA GGC GCA
GCG GTC GGG CTG AAC GGG GGG TTC GTG CAC ACA GCC CAG CTT GGA GCG AAC GAC CTA CAC
CGA ACT GAG ATA CCT ACA GCG TGA GCT ATG AGA AAG CGC CAC GCT TCC CGA AGG GAG AAA
GGC GGA CAG GTA TCC GGT AAG CGG CAG GGT CGG AAC AGG AGA GCG CAC GAG GGA GCT TCC
AGG GGG AAA CGC CTG GTA TCT TTA TAG TCC TGT CGG GTT TCG CCA CCT CTG ACT TGA GCG
TCG ATT TTT GTG ATG CTC GTC AGG GGG GCG GAG CCT ATG GAA AAA CGC CAG CAA CGC GGC
CTT TTT ACG GTT CCT GGC CTT TTG CTG GCC TTT TGC TCA CAT GTT CTT TCC TGC GTT ATC
CCC TGA TTC TGT GGA TAA CCG TAT TAC CGC TAG CCA GGA AGA GTT TGT AGA AAC GCA AAA
AGG CCA TCC GTC AGG ATG GCC TTC TGC TTA GTT TGA TGC CTG GCA GTT TAT GGC GGG CGT
CCT GCC CGC CAC CCT CCG GGC CGT TGC TTC ACA ACG TTC AAA TCC GCT CCC GGC GGA TTT
GTC CTA CTC AGG AGA GCG TTC ACC GAC AAA CAA CAG ATA AAA CGA AAG GCC CAG TCT TCC
GAC TGA GCC TTT CGT TTT ATT TGA TGC CTG GCA GTT CCC TAC TCT CGC GTT AAC GCT AGC
ATG GAT CTC GGG CCC CAA ATA ATG ATT TTA TTT TGA CTG ATA GTG ACC
TGT TCG TTG CAA CAA ATT GAT GAG CAA TGC TTT TTT ATA ATG CCA
ACT TTG TAC AAA AAA GCA GGC TCC AGG AAA ATG ATC CTT CTG CTG CTG GGT
CT CGG AAA GCA AAA TAA ACT ACG GAC CGT CAA GGG ATG AGA GCG CAA TTG CGA TCG
TAC CTA GAG CCC GGG GTT TAT TAC TAA AAT AAA ACT GAC TAT CAC TGG
ACA AGC AAC GTT GTT TAA CTA CTC GTT ACG AAA AAA TAT TAC GGT
TGA AAC ATG TTT TTT CGT CCG AGG TCC TTT TAC TAG GAA GAC GAC GAC CCA
GAG ACA TCG TGG CGG ATG TAT GGA GCG AGA CGA TTA GGA CAA TGG TCA CCG ACG ACG GTC
ACC GCT ATT CAG CAC AGA ATG GCC CAA CCT GAG TTC TGC TAT CAA TGG CCT ATT CCG CGT
CGC CAG CCC GAC TTG CCC CCC AAG CAC GTG TGT CGG GTC GAA CCT CGC TTG CTG GAT GTG
GCT TGA CTC TAT GGA TGT CGC ACT CGA TAC TCT TTC GCG GTG CGA AGG GCT TCC CTC TTT
CCG CCT GTC CAT AGG CCA TTC GCC GTC CCA GCC TTG TCC TCT CGC GTG CTC CCT CGA AGG
TCC CCC TTT GCG GAC CAT AGA AAT ATC AGG ACA GCC CAA AGC GGT GGA GAC TGA ACT CGC
AGC TAA AAA CAC TAC GAG CAG TCC CCC CGC CTC GGA TAC CTT TTT GCG GTC GTT GCG CCG
GAA AAA TGC CAA GGA CCG GAA AAC GAC CGG AAA ACG AGT GTA CAA GAA AGG ACG CAA TAG
GGG ACT AAG ACA CCT ATT GGC ATA ATG GCG ATC GGT CCT TCT CAA ACA TCT TTG CGT TTT
TCC GGT AGG CAG TCC TAC CGG AAG ACG AAT CAA ACT ACG GAC CGT CAA ATA CCG CCC GCA
GGA CGG GCG GTG GGA GGC CCG GCA ACG AAG TGT TGC AAG TTT AGG CGA GGG CCG CCT AAA
CAG GAT GAG TCC TCT CGC AAG TGG CTG TTT GTT GTC TAT TTT GCT TTC CGG GTC AGA AGG
CTG A
M I L L L L G
V L V V L M A L P P P T A G T T D W M Q
S C G T C H C Q W N S G K K S A D C K N
K A L T K I P Q D M S N E M Q V L D F A
H N Q I P E L R R E E F L L A G L P N V
H K I F L R N C T I Q E V H R E A F K G
L H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E V
L P N H L F V N L S F L S R I E F R N N
R L R Q V Q L H V F A G T M A L S A I S
L E Q N R L S H L H K E T F K D L Q K L
M H L S L Q G N A W N C S C E L Q D F R
D F A I S K R L Y T P P T D C Q E P P Q
L R G K L W S E V P S E N F A C R P R I
L G S V R S F I E A N H D N I S L P C R
I V G S P R P N V T W V Y N K R P L Q Q
Y D P R V R V L T S V E Q M P E Q P S Q
V L T S E L R I V G V R A S D K G A Y T
C V A D N R G G R A E A E F Q L L V S G
D Y A G A V S A S D G M G M G A I G A P
T I D P Q T N M F L I I C L I I T T L L
L L L L V A V L T L F W Y C R R I K T Y
Q K D T T M M S G D G L I S S K M D K T
H N G S M L E G S V I M E M Q K S L L N
E V N P V E K P P R R T D I E S V D G G
D D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R S
R H T Y V D D A Y A N S L P P D L L A F
P A R V P P T S P S M Q S S Q S N I P D
Q V I Y G I R S P P S L T S P V Y T H M
T P H G I Y G T K T M T A P H N G F M T
L Q H P K S R N L A L I A T T N S S R Q
H Q H H H Q L Q Q Q Q Q H H H H H Q Q Q
Q Q Q Q Q Q Q Q H P L A T T S P F L P A
P V V Y S P A T G V E P V Y D N L G L R
T T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T S
V S S A T N A S K I Y E P I H E L I Q Q
Q Q Q L Q Q Q Q Q Q Q Q Q R L G S M D T
E P L Y G V R Q Q G I T I L P G S S I S
G A G L G H A A Y L S P G S G A A V S P
S H A S S S G D S P K A A K I P P R P P
P K P K K K M S V T T T R S G Q G S T S
Q L F D D E G E D G T E V
W50
Start of Good Sequence (D7, pENTRK5∆3)
NheI
NheI
attL1
junction markersignal sequence
KEK5
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
1981
2041
2089
2134
1
8
28
48
68
88
108
128
148
168
188
208
228
248
268
288
308
328
348
368
388
408
428
448
468
488
508
528
548
568
588
608
628
648
668
688
708
728
748
768
788
808
828
848
GTG CTA GTG GTT CTA ATG GCC CTA CCG CCG CCC ACC GCA GGC ACC ACC GAT TGG ATG CAG
AGC TGC GGT ACA TGC CAC TGT CAG TGG AAT TCG GGC AAG AAG AGC GCC GAC TGC AAG AAC
AAG GCG CTA ACC AAA ATT CCG CAG GAC ATG AGC AAC GAG ATG CAG GTG CTG GAC TTT GCC
CAC AAT CAA ATA CCC GAG CTG CGG CGC GAA GAG TTC CTA CTG GCC GGT CTG CCC AAT GTG
CAC AAG ATC TTT TTG CGC AAC TGC ACC ATC CAG GAG GTG CAT CGC GAG GCC TTC AAG GGT
CTG CAT ATC CTA ATC GAG CTG GAC CTG TCG GGC AAT CGG ATA CGG GAA CTG CAT CCG GGC
ACT TTC GCC GGC CTG GAG AAG CTG CGC AAC gTG ATC ATC AAC AAC AAC GAG ATC GAG GTG
CTG CCC AAC CAT CTG TTC GTC AAC CTG AGC TTC CTG TCG CGC ATC GAG TTC CGG AAC AAT
CGA TTG CGC CAG GTG CAG CTG CAC GTC TTC GCT GGC ACA ATG GCG CTG AGC GCC ATT TCG
CTG GAA CAG AAC CGC CTC TCA CAT CTG CAC AAG GAG ACA TTC AAG GAT CTG CAG AAG CTG
ATG CAT CTA TCG CTG CAG GGT AAC GCA TGG AAC TGC AGC TGC GAG CTG CAG GAC TTT CGC
CAC GAT CAC CAA GAT TAC CGG GAT GGC GGC GGG TGG CGT CCG TGG TGG CTA ACC TAC GTC
TCG ACG CCA TGT ACG GTG ACA GTC ACC TTA AGC CCG TTC TTC TCG CGG CTG ACG TTC TTG
TTC CGC GAT TGG TTT TAA GGC GTC CTG TAC TCG TTG CTC TAC GTC CAC GAC CTG AAA CGG
GTG TTA GTT TAT GGG CTC GAC GCC GCG CTT CTC AAG GAT GAC CGG CCA GAC GGG TTA CAC
GTG TTC TAG AAA AAC GCG TTG ACG TGG TAG GTC CTC CAC GTA GCG CTC CGG AAG TTC CCA
GAC GTA TAG GAT TAG CTC GAC CTG GAC AGC CCG TTA GCC TAT GCC CTT GAC GTA GGC CCG
TGA AAG CGG CCG GAC CTC TTC GAC GCG TTG cAC TAG TAG TTG TTG TTG CTC TAG CTC CAC
GAC GGG TTG GTA GAC AAG CAG TTG GAC TCG AAG GAC AGC GCG TAG CTC AAG GCC TTG TTA
GCT AAC GCG GTC CAC GTC GAC GTG CAG AAG CGA CCG TGT TAC CGC GAC TCG CGG TAA AGC
GAC CTT GTC TTG GCG GAG AGT GTA GAC GTG TTC CTC TGT AAG TTC CTA GAC GTC TTC GAC
TAC GTA GAT AGC GAC GTC CCA TTG CGT ACC TTG ACG TCG ACG CTC GAC GTC CTG AAA GCG
M I L L L L G
V L V V L M A L P P P T A G T T D W M Q
S C G T C H C Q W N S G K K S A D C K N
K A L T K I P Q D M S N E M Q V L D F A
H N Q I P E L R R E E F L L A G L P N V
H K I F L R N C T I Q E V H R E A F K G
L H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E V
L P N H L F V N L S F L S R I E F R N N
R L R Q V Q L H V F A G T M A L S A I S
L E Q N R L S H L H K E T F K D L Q K L
M H L S L Q G N A W N C S C E L Q D F R
D F A I S K R L Y T P P T D C Q E P P Q
L R G K L W S E V P S E N F A C R P R I
L G S V R S F I E A N H D N I S L P C R
I V G S P R P N V T W V Y N K R P L Q Q
Y D P R V R V L T S V E Q M P E Q P S Q
V L T S E L R I V G V R A S D K G A Y T
C V A D N R G G R A E A E F Q L L V S G
D Y A G A V S A S D G M G M G A I G A P
T I D P Q T N M F L I I C L I I T T L L
L L L L V A V L T L F W Y C R R I K T Y
Q K D T T M M S G D G L I S S K M D K T
H N G S M L E G S V I M E M Q K S L L N
E V N P V E K P P R R T D I E S V D G G
D D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R S
R H T Y V D D A Y A N S L P P D L L A F
P A R V P P T S P S M Q S S Q S N I P D
Q V I Y G I R S P P S L T S P V Y T H M
T P H G I Y G T K T M T A P H N G F M T
L Q H P K S R N L A L I A T T N S S R Q
H Q H H H Q L Q Q Q Q Q H H H H H Q Q Q
Q Q Q Q Q Q Q Q H P L A T T S P F L P A
P V V Y S P A T G V E P V Y D N L G L R
T T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T S
V S S A T N A S K I Y E P I H E L I Q Q
Q Q Q L Q Q Q Q Q Q Q Q Q R L G S M D T
E P L Y G V R Q Q G I T I L P G S S I S
G A G L G H A A Y L S P G S G A A V S P
S H A S S S G D S P K A A K I P P R P P
P K P K K K M S V T T T R S G Q G S T S
Q L F D D E G E D G T E V
N FLANK
LRRs
BglII
Actual sequence is GTG - should match original wt sequenceoligo238
oligo594
C FLANK
2185
2245
2305
2365
2425
2485
2545
2605
2665
2725
2785
1
8
28
48
68
88
108
128
148
168
188
208
228
248
268
288
308
328
348
368
388
408
428
448
468
488
508
528
548
568
588
608
628
648
668
688
708
728
748
768
788
808
828
848
GAC TTT GCG ATC AGC AAA CGG CTC TAC ACA CCG CCC ACC GAT TGC CAG GAG CCG CCA CAG
CTG CGC GGC AAG CTG TGG AGC GAG GTG CCA TCG GAG AAC TTC GCC TGC CGG CCG CGC ATT
TTG GGT TCC GTG CGC TCC TTC ATC GAG GCC AAT CAC GAC AAT ATC TCG CTA CCC TGC CGC
ATT GTC GGC AGT CCG CGT CCC AAT GTC ACC TGG GTG TAC AAC AAG CGG CCA TTG CAG CAG
TAC GAC CCG CGT GTG CGT GTC CTC ACC TCC GTG GAA CAG ATG CCG GAG CAG CCC TCC CAG
GTG CTC ACC TCG GAG CTG CGC ATC GTG GGC GTA CGG GCC TCC GAC AAG GGT GCC TAC ACC
TGT GTG GCG GAT AAC CGG GGC GGA CGG GCG GAG GCC GAG TTC CAG CTG CTC GTG AGC GGT
GAC TAT GCC GGC GCG GTA TCC GCC TCC GAT GGC ATG GGC ATG GGC GCC ATT GGG GCA CCA
ACC ATT GAT CCG CAA ACG AAC ATG TTT CTC ATC ATC TGT CTA ATC ATT ACG ACG CTG CTG
CTC CTG CTG CTC GTG GCG GTG CTG ACG CTC TTC TGG TAC TGC CGT CGC ATC AAG ACC TAT
CAA AAG GAC ACC ACC ATG ATG AGC GGC GAC GGG CTG ATC TCT TCC AAG ATG GAC AAG ACG
CTG AAA CGC TAG TCG TTT GCC GAG ATG TGT GGC GGG TGG CTA ACG GTC CTC GGC GGT GTC
GAC GCG CCG TTC GAC ACC TCG CTC CAC GGT AGC CTC TTG AAG CGG ACG GCC GGC GCG TAA
AAC CCA AGG CAC GCG AGG AAG TAG CTC CGG TTA GTG CTG TTA TAG AGC GAT GGG ACG GCG
TAA CAG CCG TCA GGC GCA GGG TTA CAG TGG ACC CAC ATG TTG TTC GCC GGT AAC GTC GTC
ATG CTG GGC GCA CAC GCA CAG GAG TGG AGG CAC CTT GTC TAC GGC CTC GTC GGG AGG GTC
CAC GAG TGG AGC CTC GAC GCG TAG CAC CCG CAT GCC CGG AGG CTG TTC CCA CGG ATG TGG
ACA CAC CGC CTA TTG GCC CCG CCT GCC CGC CTC CGG CTC AAG GTC GAC GAG CAC TCG CCA
CTG ATA CGG CCG CGC CAT AGG CGG AGG CTA CCG TAC CCG TAC CCG CGG TAA CCC CGT GGT
TGG TAA CTA GGC GTT TGC TTG TAC AAA GAG TAG TAG ACA GAT TAG TAA TGC TGC GAC GAC
GAG GAC GAC GAG CAC CGC CAC GAC TGC GAG AAG ACC ATG ACG GCA GCG TAG TTC TGG ATA
GTT TTC CTG TGG TGG TAC TAC TCG CCG CTG CCC GAC TAG AGA AGG TTC TAC CTG TTC TGC
M I L L L L G
V L V V L M A L P P P T A G T T D W M Q
S C G T C H C Q W N S G K K S A D C K N
K A L T K I P Q D M S N E M Q V L D F A
H N Q I P E L R R E E F L L A G L P N V
H K I F L R N C T I Q E V H R E A F K G
L H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E V
L P N H L F V N L S F L S R I E F R N N
R L R Q V Q L H V F A G T M A L S A I S
L E Q N R L S H L H K E T F K D L Q K L
M H L S L Q G N A W N C S C E L Q D F R
D F A I S K R L Y T P P T D C Q E P P Q
L R G K L W S E V P S E N F A C R P R I
L G S V R S F I E A N H D N I S L P C R
I V G S P R P N V T W V Y N K R P L Q Q
Y D P R V R V L T S V E Q M P E Q P S Q
V L T S E L R I V G V R A S D K G A Y T
C V A D N R G G R A E A E F Q L L V S G
D Y A G A V S A S D G M G M G A I G A P
T I D P Q T N M F L I I C L I I T T L L
L L L L V A V L T L F W Y C R R I K T Y
Q K D T T M M S G D G L I S S K M D K T
H N G S M L E G S V I M E M Q K S L L N
E V N P V E K P P R R T D I E S V D G G
D D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R S
R H T Y V D D A Y A N S L P P D L L A F
P A R V P P T S P S M Q S S Q S N I P D
Q V I Y G I R S P P S L T S P V Y T H M
T P H G I Y G T K T M T A P H N G F M T
L Q H P K S R N L A L I A T T N S S R Q
H Q H H H Q L Q Q Q Q Q H H H H H Q Q Q
Q Q Q Q Q Q Q Q H P L A T T S P F L P A
P V V Y S P A T G V E P V Y D N L G L R
T T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T S
V S S A T N A S K I Y E P I H E L I Q Q
Q Q Q L Q Q Q Q Q Q Q Q Q R L G S M D T
E P L Y G V R Q Q G I T I L P G S S I S
G A G L G H A A Y L S P G S G A A V S P
S H A S S S G D S P K A A K I P P R P P
P K P K K K M S V T T T R S G Q G S T S
Q L F D D E G E D G T E V
Ig
end Ig?
Tm
IC
oligo745 apK5_seq_int_5'
2845
2905
2965
3025
3085
3145
3205
3265
3325
3385
3445
1
8
28
48
68
88
108
128
148
168
188
208
228
248
268
288
308
328
348
368
388
408
428
448
468
488
508
528
548
568
588
608
628
648
668
688
708
728
748
768
788
808
828
848
CAC AAC GGC TCC ATG CTC GAG GGT TCC GTC ATC ATG GAG ATG CAG AAG AGC CTG CTC AAC
GAG GTC AAT CCA GTC GAG AAG CCG CCA CGG CGC ACG GAC ATC GAG AGC GTG GAT GGT GGC
GAT GAC GTG CTC GAG ATC AAG AAG ACG CTG CTC GAC GAC ACC GTC TAT GTG GCC AAT CAC
TCG CGC GAC GAA GAA GCC GTC TCA GTG GCC ATG TCG GAT ACG ACG ACC ACG CCC CGA TCT
CGA CAC ACC TAC GTG GAT GAT GCG TAT GCC AAT AGC TTG CCA CCG GAT CTG CTG GCC TTT
CCC GCT CGC GTG CCG CCC ACC TCG CCC TCG ATG CAA TCG TCG CAG TCG AAC ATA CCC GAC
CAG GTG ATC TAC GGC ATC CGT TCG CCA CCG TCG CTA ACC AGT CCG GTC TAC ACG CAT ATG
ACG CCG CAC GGC ATC TAC GGC ACC AAG ACG ATG ACG GCT CCG CAT AAC GGC TTT ATG ACG
CTG CAG CAT CCC AAG TCG CGC AAC CTG GCG CTC ATT GCC ACC ACC AAC AGC AGT CGC CAG
CAC CAG CAC CAC CAT CAG CTG CAG CAG CAG CAG CAG CAC CAC CAC CAC CAC CAG CAG CAA
GTG TTG CCG AGG TAC GAG CTC CCA AGG CAG TAG TAC CTC TAC GTC TTC TCG GAC GAG TTG
CTC CAG TTA GGT CAG CTC TTC GGC GGT GCC GCG TGC CTG TAG CTC TCG CAC CTA CCA CCG
CTA CTG CAC GAG CTC TAG TTC TTC TGC GAC GAG CTG CTG TGG CAG ATA CAC CGG TTA GTG
AGC GCG CTG CTT CTT CGG CAG AGT CAC CGG TAC AGC CTA TGC TGC TGG TGC GGG GCT AGA
GCT GTG TGG ATG CAC CTA CTA CGC ATA CGG TTA TCG AAC GGT GGC CTA GAC GAC CGG AAA
GGG CGA GCG CAC GGC GGG TGG AGC GGG AGC TAC GTT AGC AGC GTC AGC TTG TAT GGG CTG
GTC CAC TAG ATG CCG TAG GCA AGC GGT GGC AGC GAT TGG TCA GGC CAG ATG TGC GTA TAC
TGC GGC GTG CCG TAG ATG CCG TGG TTC TGC TAC TGC CGA GGC GTA TTG CCG AAA TAC TGC
GAC GTC GTA GGG TTC AGC GCG TTG GAC CGC GAG TAA CGG TGG TGG TTG TCG TCA GCG GTC
GTG GTC GTG GTG GTA GTC GAC GTC GTC GTC GTC GTC GTG GTG GTG GTG GTG GTC GTC GTT
M I L L L L G
V L V V L M A L P P P T A G T T D W M Q
S C G T C H C Q W N S G K K S A D C K N
K A L T K I P Q D M S N E M Q V L D F A
H N Q I P E L R R E E F L L A G L P N V
H K I F L R N C T I Q E V H R E A F K G
L H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E V
L P N H L F V N L S F L S R I E F R N N
R L R Q V Q L H V F A G T M A L S A I S
L E Q N R L S H L H K E T F K D L Q K L
M H L S L Q G N A W N C S C E L Q D F R
D F A I S K R L Y T P P T D C Q E P P Q
L R G K L W S E V P S E N F A C R P R I
L G S V R S F I E A N H D N I S L P C R
I V G S P R P N V T W V Y N K R P L Q Q
Y D P R V R V L T S V E Q M P E Q P S Q
V L T S E L R I V G V R A S D K G A Y T
C V A D N R G G R A E A E F Q L L V S G
D Y A G A V S A S D G M G M G A I G A P
T I D P Q T N M F L I I C L I I T T L L
L L L L V A V L T L F W Y C R R I K T Y
Q K D T T M M S G D G L I S S K M D K T
H N G S M L E G S V I M E M Q K S L L N
E V N P V E K P P R R T D I E S V D G G
D D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R S
R H T Y V D D A Y A N S L P P D L L A F
P A R V P P T S P S M Q S S Q S N I P D
Q V I Y G I R S P P S L T S P V Y T H M
T P H G I Y G T K T M T A P H N G F M T
L Q H P K S R N L A L I A T T N S S R Q
H Q H H H Q L Q Q Q Q Q H H H H H Q Q Q
Q Q Q Q Q Q Q Q H P L A T T S P F L P A
P V V Y S P A T G V E P V Y D N L G L R
T T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T S
V S S A T N A S K I Y E P I H E L I Q Q
Q Q Q L Q Q Q Q Q Q Q Q Q R L G S M D T
E P L Y G V R Q Q G I T I L P G S S I S
G A G L G H A A Y L S P G S G A A V S P
S H A S S S G D S P K A A K I P P R P P
P K P K K K M S V T T T R S G Q G S T S
Q L F D D E G E D G T E V
IC1 modified oligo746 apK5_seq_int_3'
∆1 ConfirmedIC1
oligo240
∆234 ConfirmedIC2 modified
IC2
3505
3565
3625
3685
3745
3805
3865
3925
3985
4045
1
8
28
48
68
88
108
128
148
168
188
208
228
248
268
288
308
328
348
368
388
408
428
448
468
488
508
528
548
568
588
608
628
648
668
688
708
728
748
768
788
808
828
848
CAA CAA CAG CAG CAG CAG CAG CAA CAT CCG CTG GCC ACC ACA TCG CCC TTC CTG CCC GCA
CCC GTC GTC TAT TCG CCG GCC ACG GGT GTG GAG CCA GTG TAC GAC AAC TTG GGA TTG CGA
ACC ACT GCC GGC GGC AAC TCC ACC CTC AAT CTG ACC AAG ATC GCC GGC TCA CAG GGG GGC
GCT GGT CAG CAG TAC TCG ATG CGG GAC CGA CCA CTT CCG GCC ACG CCC AGC CTG ACA TCG
GTG TCC TCG GCG ACC AAT GCC AGT AAG ATT TAC GAG CCC ATA CAC GAG CTG ATT CAG CAG
CAA CAG CAG TTG CAA CAA CAA CAA CAG CAG CAG CAG CAG CGA CTG GGC TCC ATG GAC ACG
GAA CCC CTG TAC GGA GTT CGG CAA CAG GGG ATC ACG ATA CTG CCC GGC TCG AGC ATT AGC
GGT GCC GGA CTG GGC CAC GCC GCC TAC CTT TCA CCC GGC TCG GGT GCC GCC GTA TCG CCA
AGC CAC GCC AGC AGC AGC GGT GAC TCT CCG AAG GCC GCC AAG ATC CCA CCA CGC CCA CCA
CCG AAG CCC AAG AAG AAG ATG TCC GTG ACG ACG ACG CGC AGC GGC CAG GGC AGC ACC AGC
CAG CTC TTC GAC GAC GAG GGC GAG GAT GGC ACC GAG GTC GAC CCA GCT TTC TTG
GTT GTT GTC GTC GTC GTC GTC GTT GTA GGC GAC CGG TGG TGT AGC GGG AAG GAC GGG CGT
GGG CAG CAG ATA AGC GGC CGG TGC CCA CAC CTC GGT CAC ATG CTG TTG AAC CCT AAC GCT
TGG TGA CGG CCG CCG TTG AGG TGG GAG TTA GAC TGG TTC TAG CGG CCG AGT GTC CCC CCG
CGA CCA GTC GTC ATG AGC TAC GCC CTG GCT GGT GAA GGC CGG TGC GGG TCG GAC TGT AGC
CAC AGG AGC CGC TGG TTA CGG TCA TTC TAA ATG CTC GGG TAT GTG CTC GAC TAA GTC GTC
GTT GTC GTC AAC GTT GTT GTT GTT GTC GTC GTC GTC GTC GCT GAC CCG AGG TAC CTG TGC
CTT GGG GAC ATG CCT CAA GCC GTT GTC CCC TAG TGC TAT GAC GGG CCG AGC TCG TAA TCG
CCA CGG CCT GAC CCG GTG CGG CGG ATG GAA AGT GGG CCG AGC CCA CGG CGG CAT AGC GGT
TCG GTG CGG TCG TCG TCG CCA CTG AGA GGC TTC CGG CGG TTC TAG GGT GGT GCG GGT GGT
GGC TTC GGG TTC TTC TTC TAC AGG CAC TGC TGC TGC GCG TCG CCG GTC CCG TCG TGG TCG
GTC GAG AAG CTG CTG CTC CCG CTC CTA CCG TGG CTC CAG CTG GGT CGA AAG AAC
M I L L L L G
V L V V L M A L P P P T A G T T D W M Q
S C G T C H C Q W N S G K K S A D C K N
K A L T K I P Q D M S N E M Q V L D F A
H N Q I P E L R R E E F L L A G L P N V
H K I F L R N C T I Q E V H R E A F K G
L H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E V
L P N H L F V N L S F L S R I E F R N N
R L R Q V Q L H V F A G T M A L S A I S
L E Q N R L S H L H K E T F K D L Q K L
M H L S L Q G N A W N C S C E L Q D F R
D F A I S K R L Y T P P T D C Q E P P Q
L R G K L W S E V P S E N F A C R P R I
L G S V R S F I E A N H D N I S L P C R
I V G S P R P N V T W V Y N K R P L Q Q
Y D P R V R V L T S V E Q M P E Q P S Q
V L T S E L R I V G V R A S D K G A Y T
C V A D N R G G R A E A E F Q L L V S G
D Y A G A V S A S D G M G M G A I G A P
T I D P Q T N M F L I I C L I I T T L L
L L L L V A V L T L F W Y C R R I K T Y
Q K D T T M M S G D G L I S S K M D K T
H N G S M L E G S V I M E M Q K S L L N
E V N P V E K P P R R T D I E S V D G G
D D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R S
R H T Y V D D A Y A N S L P P D L L A F
P A R V P P T S P S M Q S S Q S N I P D
Q V I Y G I R S P P S L T S P V Y T H M
T P H G I Y G T K T M T A P H N G F M T
L Q H P K S R N L A L I A T T N S S R Q
H Q H H H Q L Q Q Q Q Q H H H H H Q Q Q
Q Q Q Q Q Q Q Q H P L A T T S P F L P A
P V V Y S P A T G V E P V Y D N L G L R
T T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T S
V S S A T N A S K I Y E P I H E L I Q Q
Q Q Q L Q Q Q Q Q Q Q Q Q R L G S M D T
E P L Y G V R Q Q G I T I L P G S S I S
G A G L G H A A Y L S P G S G A A V S P
S H A S S S G D S P K A A K I P P R P P
P K P K K K M S V T T T R S G Q G S T S
Q L F D D E G E D G T E V
∆ IC3CLIC4
IC5
IC6 (PDZ) attB2attL2
4105
4165
4225
4285
4345
4405
4465
4525
4585
4645
4705
1
8
28
48
68
88
108
128
148
168
188
208
228
248
268
288
308
328
348
368
388
408
428
448
468
488
508
528
548
568
588
608
628
648
668
688
708
728
748
768
788
808
828
848
TAC AAA GTT GGC ATT ATA AGA AAG CAT TGC TTA TCA ATT TGT TGC
AAC GAA CAG GTC ACT ATC AGT CAA AAT AAA ATC ATT ATT TG
ATG TTT CAA CCG TAA TAT TCT TTC GTA ACG AAT AGT TAA ACA ACG
TTG CTT GTC CAG TGA TAG TCA GTT TTA TTT TAG TAA TAA AC
End of Good Sequence (D7, pENTRK5∆3)
4759
4804
GGCCAGACCCACGTAGTCCAGCGGCAGATCGGCGGCGGAGAAGTTAAGCGTCTCCAGGATGACCTTGCCCGAACTGGG
GCACGTGGTGTTCGACGATGTGCAGCTAATTTCGCCCGGCTCCACGTCCGCCCATTGGTTAATCAGCAGACCCTCGTT
GGCGTAACGGAACCATGAGAGGTACGACAACCATTTGAGGTATACTGGCACCGAGCCCGAGTTCAAGAAGAAGGCGTT
TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT
ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT
GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGT
TCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA
GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG
CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT
GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT
TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC
TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAA
TTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGA
GGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACG
GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAAT
AAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTG
CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC
ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTG
CAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT
GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC
ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAG
AACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG
TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAAC
AGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATA
CCGGTCTGGGTGCATCAGGTCGCCGTCTAGCCGCCGCCTCTTCAATTCGCAGAGGTCCTACTGGAACGGGCTTGACCC
CGTGCACCACAAGCTGCTACACGTCGATTAAAGCGGGCCGAGGTGCAGGCGGGTAACCAATTAGTCGTCTGGGAGCAA
CCGCATTGCCTTGGTACTCTCCATGCTGTTGGTAAACTCCATATGACCGTGGCTCGGGCTCAAGTTCTTCTTCCGCAA
AAAGGTATCCGAGGCGGGGGGACTGCTCGTAGTGTTTTTAGCTGCGAGTTCAGTCTCCACCGCTTTGGGCTGTCCTGA
TATTTCTATGGTCCGCAAAGGGGGACCTTCGAGGGAGCACGCGAGAGGACAAGGCTGGGACGGCGAATGGCCTATGGA
CAGGCGGAAAGAGGGAAGCCCTTCGCACCGCGAAAGAGTTACGAGTGCGACATCCATAGAGTCAAGCCACATCCAGCA
AGCGAGGTTCGACCCGACACACGTGCTTGGGGGGCAAGTCGGGCTGGCGACGCGGAATAGGCCATTGATAGCAGAACT
CAGGTTGGGCCATTCTGTGCTGAATAGCGGTGACCGTCGTCGGTGACCATTGTCCTAATCGTCTCGCTCCATACATCC
GCCACGATGTCTCAAGAACTTCACCACCGGATTGATGCCGATGTGATCTTCCTGTCATAAACCATAGACGCGAGACGA
CTTCGGTCAATGGAAGCCTTTTTCTCAACCATCGAGAACTAGGCCGTTTGTTTGGTGGCGACCATCGCCACCAAAAAA
ACAAACGTTCGTCGTCTAATGCGCGTCTTTTTTTCCTAGAGTTCTTCTAGGAAACTAGAAAAGATGCCCCAGACTGCG
AGTCACCTTGCTTTTGAGTGCAATTCCCTAAAACCAGTACTCTAATAGTTTTTCCTAGAAGTGGATCTAGGAAAATTT
AATTTTTACTTCAAAATTTAGTTAGATTTCATATATACTCATTTGAACCAGACTGTCAATGGTTACGAATTAGTCACT
CCGTGGATAGAGTCGCTAGACAGATAAAGCAAGTAGGTATCAACGGACTGAGGGGCAGCACATCTATTGATGCTATGC
CCTCCCGAATGGTAGACCGGGGTCACGACGTTACTATGGCGCTCTGGGTGCGAGTGGCCGAGGTCTAAATAGTCGTTA
TTTGGTCGGTCGGCCTTCCCGGCTCGCGTCTTCACCAGGACGTTGAAATAGGCGGAGGTAGGTCAGATAATTAACAAC
GGCCCTTCGATCTCATTCATCAAGCGGTCAATTATCAAACGCGTTGCAACAACGGTAACGATGTCCGTAGCACCACAG
TGCGAGCAGCAAACCATACCGAAGTAAGTCGAGGCCAAGGGTTGCTAGTTCCGCTCAATGTACTAGGGGGTACAACAC
GTTTTTTCGCCAATCGAGGAAGCCAGGAGGCTAGCAACAGTCTTCATTCAACCGGCGTCACAATAGTGAGTACCAATA
CCGTCGTGACGTATTAAGAGAATGACAGTACGGTAGGCATTCTACGAAAAGACACTGACCACTCATGAGTTGGTTCAG
TAAGACTCTTATCACATACGCCGCTGGCTCAACGAGAACGGGCCGCAGTTGTGCCCTATTATGGCGCGGTGTATCGTC
TTGAAATTTTCACGAGTAGTAACCTTTTGCAAGAAGCCCCGCTTTTGAGAGTTCCTAGAATGGCGACAACTCTAGGTC
AAGCTACATTGGGTGAGCACGTGGGTTGACTAGAAGTCGTAGAAAATGAAAGTGGTCGCAAAGACCCACTCGTTTTTG
TCCTTCCGTTTTACGGCGTTTTTTCCCTTATTCCCGCTGTGCCTTTACAACTTATGAGTATGAGAAGGAAAAAGTTAT
1
79
157
235
313
391
469
547
625
703
781
859
937
1015
1093
1171
1249
1327
1405
1483
1561
1639
1717
1795
TTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGG
GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAA
TAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGA
GACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTG
TCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACCG
AATCGCGCGGAACTAACGACAGTCGCTCCAAGGTCGTCGAACAAAAGGTGAATGTGTTGCGGAGAGCGGGTGGGAGAC
AGCGAAAGAGCAACTACGAAACGTGGTGTGGTGGAGGTGAATTATGAAGAGGGCGCGCGATTTGAAAAGTATGTATAT
AAAAAATATATCCCGGTGTTTTATGTAGCGATAAACGAGTTTTTGATGTAAGGTATGCAGGTGTGTAAGTCTTTTGGT
TAGAAGACAAATCCAAAGTCTACTTGTGGGGATGTTCGAAGGGGAAATACTTGTATTCTATAGGTCATATCTTGTTTT
TATTGGCACAAATATAATTACATTAGCTTTTTGAGGGGGCAATAAACAGTAAACACGATGGTAATAATGGTAAAAAAA
AAAACAAGCAGTTATTTCGGATATATGTCGGCTACTCCTTGCGTCGGGCCCGAAGTCTTAGAGCCAGATATGCGAGCA
CCCGGAAGCTCACGATGAGAATGGCCAGACCATGATGAAATAACATAAGGTGGTCCCGTCGGCAAGAGACATCCACTT
AACGTATGCTTGCAATAAGTGCGAGTGAAAGGAATAGTATTCTGAGTGTCGTATTGAGTCTGAGTGAGACAGCGATAT
GATTGTTGATTAACCCTTAGCATGTCCGTGGGGTTTGAATTAACTCATAATATTAATTAGACGAAATTATTTTTAAAG
TTTTATTTTTAATAATTTGCGAGTACGCAAAGCTTCTGCATGAGCTCGGATCCAAGCTTGCATGCCTGCAGGTCGGAG
TACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTA
CTGTCCTCCGAGCGGAGACTCTAGCGAGCGCCGGAGTATAAATAGAGGCGCTTCGTCTACGGAGCGACAATTCAATTC
AAACAAGCAAAGTGAACACGTCGCTAAGCGAAAGCTAAGCAAATAAACAAGCGCAGCTGAACAAGCTAAACAATCTGC
AGTAAAGTGCAAGTTAAAGTGAATCAATTAAAAGTAACCAGCAACCAAGTAAATCAACTGCAACTACTGAAATCTGCC
AATAACTTCGTAAATAGTCCCAATAACAGAGTACTCGCCTATGTATAAACTTACATAAATCTTTTTATTTGTTTATCC
CCAAGGCGCGTGTAAAGGGGCTTTTCACGGTGGACTGCAGATTCTTTGGTAATAATAGTACTGTAATTGGATATTTTT
ATCCGCATAGTGCTCCGGGAAAGCAGAGCGCGCAAAGCCACTACTGCCACTTTTGGAGACTGTGTACGTCGAGGGCCT
CTGCCAGTGTCGAACAGACATTCGCCTACGGCCCTCGTCTGTTCGGGCAGTCCCGCGCAGTCGCCCACAACCGCCCAC
AGCCCCGACCGAATTGATACGCCGTAGTCTCGTCTAACATGACTCTCACGTGGTATACGCCACACTTTATGGCGTGGC
TTAGCGCGCCTTGATTGCTGTCAGCGAGGTTCCAGCAGCTTGTTTTCCACTTACACAACGCCTCTCGCCCACCCTCTG
TCGCTTTCTCGTTGATGCTTTGCACCACACCACCTCCACTTAATACTTCTCCCGCGCGCTAAACTTTTCATACATATA
TTTTTTATATAGGGCCACAAAATACATCGCTATTTGCTCAAAAACTACATTCCATACGTCCACACATTCAGAAAACCA
ATCTTCTGTTTAGGTTTCAGATGAACACCCCTACAAGCTTCCCCTTTATGAACATAAGATATCCAGTATAGAACAAAA
ATAACCGTGTTTATATTAATGTAATCGAAAAACTCCCCCGTTATTTGTCATTTGTGCTACCATTATTACCATTTTTTT
TTTTGTTCGTCAATAAAGCCTATATACAGCCGATGAGGAACGCAGCCCGGGCTTCAGAATCTCGGTCTATACGCTCGT
GGGCCTTCGAGTGCTACTCTTACCGGTCTGGTACTACTTTATTGTATTCCACCAGGGCAGCCGTTCTCTGTAGGTGAA
TTGCATACGAACGTTATTCACGCTCACTTTCCTTATCATAAGACTCACAGCATAACTCAGACTCACTCTGTCGCTATA
CTAACAACTAATTGGGAATCGTACAGGCACCCCAAACTTAATTGAGTATTATAATTAATCTGCTTTAATAAAAATTTC
AAAATAAAAATTATTAAACGCTCATGCGTTTCGAAGACGTACTCGAGCCTAGGTTCGAACGTACGGACGTCCAGCCTC
ATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCAT
GACAGGAGGCTCGCCTCTGAGATCGCTCGCGGCCTCATATTTATCTCCGCGAAGCAGATGCCTCGCTGTTAAGTTAAG
TTTGTTCGTTTCACTTGTGCAGCGATTCGCTTTCGATTCGTTTATTTGTTCGCGTCGACTTGTTCGATTTGTTAGACG
TCATTTCACGTTCAATTTCACTTAGTTAATTTTCATTGGTCGTTGGTTCATTTAGTTGACGTTGATGACTTTAGACGG
3 ' P
UAS sites
1873
1951
2029
2107
2185
2263
2341
2419
2497
2575
2653
2731
2809
2887
2965
3043
3121
3199
3277
AAGAAGTAATTATTGAATACAAGAAGAGAACTCTGAATAGGGAATTGGGGAATTCGTTAACAGATCTGACAAGTTTGT
ACAAAAAAGCAGGCTCCAGGAAA ATG ATC CTT CTG CTG CTG GGT GTG CTA GTG GTT CTA ATG
GCC CTA CCG CCG CCC ACC GCA GGC ACC ACC GAT TGG ATG CAG AGC TGC GGT ACA TGC
CAC TGT CAG TGG AAT TCG GGC AAG AAG AGC GCC GAC TGC AAG AAC AAG GCG CTA ACC
AAA ATT CCG CAG GAC ATG AGC AAC GAG ATG CAG GTG CTG GAC TTT GCC CAC AAT CAA
ATA CCC GAG CTG CGG CGC GAA GAG TTC CTA CTG GCC GGT CTG CCC AAT GTG CAC AAG
ATC TTT TTG CGC AAC TGC ACC ATC CAG GAG GTG CAT CGC GAG GCC TTC AAG GGT CTG
CAT ATC CTA ATC GAG CTG GAC CTG TCG GGC AAT CGG ATA CGG GAA CTG CAT CCG GGC
TTCTTCATTAATAACTTATGTTCTTCTCTTGAGACTTATCCCTTAACCCCTTAAGCAATTGTCTAGACTGTTCAAACA
TGTTTTTTCGTCCGAGGTCCTTT TAC TAG GAA GAC GAC GAC CCA CAC GAT CAC CAA GAT TAC
CGG GAT GGC GGC GGG TGG CGT CCG TGG TGG CTA ACC TAC GTC TCG ACG CCA TGT ACG
GTG ACA GTC ACC TTA AGC CCG TTC TTC TCG CGG CTG ACG TTC TTG TTC CGC GAT TGG
TTT TAA GGC GTC CTG TAC TCG TTG CTC TAC GTC CAC GAC CTG AAA CGG GTG TTA GTT
TAT GGG CTC GAC GCC GCG CTT CTC AAG GAT GAC CGG CCA GAC GGG TTA CAC GTG TTC
TAG AAA AAC GCG TTG ACG TGG TAG GTC CTC CAC GTA GCG CTC CGG AAG TTC CCA GAC
GTA TAG GAT TAG CTC GAC CTG GAC AGC CCG TTA GCC TAT GCC CTT GAC GTA GGC CCG
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P Y G I R
S P P S L T S P V Y T H M T P H G I Y
G T K T M T A P H N G F M T L Q H P K
S R N L A L I A T T N S S R Q H Q H H
H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V
V Y S P A T G V V M K Q G Y M T I P R
K P R A P S W A P S T S G A A G H G S
I Q L S E F Q S P T S P N P S E T G T
A T T A E L Q A E P V Y D N L G L R T
T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T
S V S S A T N A S K I Y E P I H E L I
Q Q Q Q Q L Q Q Q Q Q Q Q Q Q R L G S
M D T E P L Y G V R Q Q G I T I L P G
S S I S G A G L G H A A Y L S P G S G
A A V S P S H A S S S G D S P K A A K
I P P R P P P K P K K K M S V T T T R
S G Q G S T S Q L F D D E G E D G T E
V D P A F L Y K V V V P R A R D P P V
A T M V S K G E E L F T G V V P I L V
E L D G D V N G H K F S V S G E G E G
D A T Y G K L T L K F I C T T G K L P
V P W P T L V T T L T Y G V Q C F S R
Y P D H M K Q H D F F K S A M P E G Y
V Q E R T I F F K D D G N Y K T R A E
V K F E G D T L V N R I E L K G I D F
K E D G N I L G H K L E Y N Y N S H N
V Y I M A D K Q K N G I K V N F K I R
H N I E D G S V Q L A D H Y Q Q N T P
I G D G P V L L P D N H Y L S T Q S A
L S K D P N E K R D H M V L L E F V T
A A G I T L G M D E L Y K •
EcoR1
P o l y l i n k e r
HpaI BglII attB1
signal sequence
KEK5
N FLANK
LRRs
BglII
3355
3433
3495
3552
3609
3666
3723
3780
1
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
1135
ACT TTC GCC GGC CTG GAG AAG CTG CGC AAC gTG ATC ATC AAC AAC AAC GAG ATC GAG
GTG CTG CCC AAC CAT CTG TTC GTC AAC CTG AGC TTC CTG TCG CGC ATC GAG TTC CGG
AAC AAT CGA TTG CGC CAG GTG CAG CTG CAC GTC TTC GCT GGC ACA ATG GCG CTG AGC
GCC ATT TCG CTG GAA CAG AAC CGC CTC TCA CAT CTG CAC AAG GAG ACA TTC AAG GAT
CTG CAG AAG CTG ATG CAT CTA TCG CTG CAG GGT AAC GCA TGG AAC TGC AGC TGC GAG
CTG CAG GAC TTT CGC GAC TTT GCG ATC AGC AAA CGG CTC TAC ACA CCG CCC ACC GAT
TGC CAG GAG CCG CCA CAG CTG CGC GGC AAG CTG TGG AGC GAG GTG CCA TCG GAG AAC
TTC GCC TGC CGG CCG CGC ATT TTG GGT TCC GTG CGC TCC TTC ATC GAG GCC AAT CAC
TGA AAG CGG CCG GAC CTC TTC GAC GCG TTG cAC TAG TAG TTG TTG TTG CTC TAG CTC
CAC GAC GGG TTG GTA GAC AAG CAG TTG GAC TCG AAG GAC AGC GCG TAG CTC AAG GCC
TTG TTA GCT AAC GCG GTC CAC GTC GAC GTG CAG AAG CGA CCG TGT TAC CGC GAC TCG
CGG TAA AGC GAC CTT GTC TTG GCG GAG AGT GTA GAC GTG TTC CTC TGT AAG TTC CTA
GAC GTC TTC GAC TAC GTA GAT AGC GAC GTC CCA TTG CGT ACC TTG ACG TCG ACG CTC
GAC GTC CTG AAA GCG CTG AAA CGC TAG TCG TTT GCC GAG ATG TGT GGC GGG TGG CTA
ACG GTC CTC GGC GGT GTC GAC GCG CCG TTC GAC ACC TCG CTC CAC GGT AGC CTC TTG
AAG CGG ACG GCC GGC GCG TAA AAC CCA AGG CAC GCG AGG AAG TAG CTC CGG TTA GTG
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P Y G I R
S P P S L T S P V Y T H M T P H G I Y
G T K T M T A P H N G F M T L Q H P K
S R N L A L I A T T N S S R Q H Q H H
H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V
V Y S P A T G V V M K Q G Y M T I P R
K P R A P S W A P S T S G A A G H G S
I Q L S E F Q S P T S P N P S E T G T
A T T A E L Q A E P V Y D N L G L R T
T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T
S V S S A T N A S K I Y E P I H E L I
Q Q Q Q Q L Q Q Q Q Q Q Q Q Q R L G S
M D T E P L Y G V R Q Q G I T I L P G
S S I S G A G L G H A A Y L S P G S G
A A V S P S H A S S S G D S P K A A K
I P P R P P P K P K K K M S V T T T R
S G Q G S T S Q L F D D E G E D G T E
V D P A F L Y K V V V P R A R D P P V
A T M V S K G E E L F T G V V P I L V
E L D G D V N G H K F S V S G E G E G
D A T Y G K L T L K F I C T T G K L P
V P W P T L V T T L T Y G V Q C F S R
Y P D H M K Q H D F F K S A M P E G Y
V Q E R T I F F K D D G N Y K T R A E
V K F E G D T L V N R I E L K G I D F
K E D G N I L G H K L E Y N Y N S H N
V Y I M A D K Q K N G I K V N F K I R
H N I E D G S V Q L A D H Y Q Q N T P
I G D G P V L L P D N H Y L S T Q S A
L S K D P N E K R D H M V L L E F V T
A A G I T L G M D E L Y K •
Actual sequence is GTG - should match original wt sequence
oligo238
oligo594
C FLANK
Ig
3837
3894
3951
4008
4065
4122
4179
4236
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
1135
GAC AAT ATC TCG CTA CCC TGC CGC ATT GTC GGC AGT CCG CGT CCC AAT GTC ACC TGG
GTG TAC AAC AAG CGG CCA TTG CAG CAG TAC GAC CCG CGT GTG CGT GTC CTC ACC TCC
GTG GAA CAG ATG CCG GAG CAG CCC TCC CAG GTG CTC ACC TCG GAG CTG CGC ATC GTG
GGC GTA CGG GCC TCC GAC AAG GGT GCC TAC ACC TGT GTG GCG GAT AAC CGG GGC GGA
CGG GCG GAG GCC GAG TTC CAG CTG CTC GTG AGC GGT GAC TAT GCC GGC GCG GTA TCC
GCC TCC GAT GGC ATG GGC ATG GGC GCC ATT GGG GCA CCA ACC ATT GAT CCG CAA ACG
AAC ATG TTT CTC ATC ATC TGT CTA ATC ATT ACG ACG CTG CTG CTC CTG CTG CTC GTG
GCG GTG CTG ACG CTC TTC TGG TAC TGC CGT CGC ATC AAG ACC TAT CAA AAG GAC ACC
CTG TTA TAG AGC GAT GGG ACG GCG TAA CAG CCG TCA GGC GCA GGG TTA CAG TGG ACC
CAC ATG TTG TTC GCC GGT AAC GTC GTC ATG CTG GGC GCA CAC GCA CAG GAG TGG AGG
CAC CTT GTC TAC GGC CTC GTC GGG AGG GTC CAC GAG TGG AGC CTC GAC GCG TAG CAC
CCG CAT GCC CGG AGG CTG TTC CCA CGG ATG TGG ACA CAC CGC CTA TTG GCC CCG CCT
GCC CGC CTC CGG CTC AAG GTC GAC GAG CAC TCG CCA CTG ATA CGG CCG CGC CAT AGG
CGG AGG CTA CCG TAC CCG TAC CCG CGG TAA CCC CGT GGT TGG TAA CTA GGC GTT TGC
TTG TAC AAA GAG TAG TAG ACA GAT TAG TAA TGC TGC GAC GAC GAG GAC GAC GAG CAC
CGC CAC GAC TGC GAG AAG ACC ATG ACG GCA GCG TAG TTC TGG ATA GTT TTC CTG TGG
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P Y G I R
S P P S L T S P V Y T H M T P H G I Y
G T K T M T A P H N G F M T L Q H P K
S R N L A L I A T T N S S R Q H Q H H
H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V
V Y S P A T G V V M K Q G Y M T I P R
K P R A P S W A P S T S G A A G H G S
I Q L S E F Q S P T S P N P S E T G T
A T T A E L Q A E P V Y D N L G L R T
T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T
S V S S A T N A S K I Y E P I H E L I
Q Q Q Q Q L Q Q Q Q Q Q Q Q Q R L G S
M D T E P L Y G V R Q Q G I T I L P G
S S I S G A G L G H A A Y L S P G S G
A A V S P S H A S S S G D S P K A A K
I P P R P P P K P K K K M S V T T T R
S G Q G S T S Q L F D D E G E D G T E
V D P A F L Y K V V V P R A R D P P V
A T M V S K G E E L F T G V V P I L V
E L D G D V N G H K F S V S G E G E G
D A T Y G K L T L K F I C T T G K L P
V P W P T L V T T L T Y G V Q C F S R
Y P D H M K Q H D F F K S A M P E G Y
V Q E R T I F F K D D G N Y K T R A E
V K F E G D T L V N R I E L K G I D F
K E D G N I L G H K L E Y N Y N S H N
V Y I M A D K Q K N G I K V N F K I R
H N I E D G S V Q L A D H Y Q Q N T P
I G D G P V L L P D N H Y L S T Q S A
L S K D P N E K R D H M V L L E F V T
A A G I T L G M D E L Y K •
end Ig?
Tm
IC ∆123 Confirmed∆1235 Confirmed∆1234 Confirmed
oligo745 apK5_seq_int_5'
4293
4350
4407
4464
4521
4578
4635
4692
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
1135
ACC ATG ATG AGC GGC GAC GGG CTG ATC TCT TCC AAG ATG GAC AAG ACG CAC AAC GGC
TCC ATG CTC GAG GGT TCC GTC ATC ATG GAG ATG CAG AAG AGC CTG CTC AAC GAG GTC
AAT CCA GTC GAG AAG CCG CCA CGG CGC ACG GAC ATC GAG AGC GTG GAT GGT GGC GAT
GAC GTG CTC GAG ATC AAG AAG ACG CTG CTC GAC GAC ACC GTC TAT GTG GCC AAT CAC
TCG CGC GAC GAA GAA GCC GTC TCA GTG GCC ATG TCG GAT ACG ACG ACC ACG CCC CGA
TCT CGA CAC ACC TAC GTG GAT GAT GCG TAT GCC AAT AGC TTG CCA TAC GGC ATC CGT
TCG CCA CCG TCG CTA ACC AGT CCG GTC TAC ACG CAT ATG ACG CCG CAC GGC ATC TAC
GGC ACC AAG ACG ATG ACG GCT CCG CAT AAC GGC TTT ATG ACG CTG CAG CAT CCC AAG
TGG TAC TAC TCG CCG CTG CCC GAC TAG AGA AGG TTC TAC CTG TTC TGC GTG TTG CCG
AGG TAC GAG CTC CCA AGG CAG TAG TAC CTC TAC GTC TTC TCG GAC GAG TTG CTC CAG
TTA GGT CAG CTC TTC GGC GGT GCC GCG TGC CTG TAG CTC TCG CAC CTA CCA CCG CTA
CTG CAC GAG CTC TAG TTC TTC TGC GAC GAG CTG CTG TGG CAG ATA CAC CGG TTA GTG
AGC GCG CTG CTT CTT CGG CAG AGT CAC CGG TAC AGC CTA TGC TGC TGG TGC GGG GCT
AGA GCT GTG TGG ATG CAC CTA CTA CGC ATA CGG TTA TCG AAC GGT ATG CCG TAG GCA
AGC GGT GGC AGC GAT TGG TCA GGC CAG ATG TGC GTA TAC TGC GGC GTG CCG TAG ATG
CCG TGG TTC TGC TAC TGC CGA GGC GTA TTG CCG AAA TAC TGC GAC GTC GTA GGG TTC
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P Y G I R
S P P S L T S P V Y T H M T P H G I Y
G T K T M T A P H N G F M T L Q H P K
S R N L A L I A T T N S S R Q H Q H H
H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V
V Y S P A T G V V M K Q G Y M T I P R
K P R A P S W A P S T S G A A G H G S
I Q L S E F Q S P T S P N P S E T G T
A T T A E L Q A E P V Y D N L G L R T
T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T
S V S S A T N A S K I Y E P I H E L I
Q Q Q Q Q L Q Q Q Q Q Q Q Q Q R L G S
M D T E P L Y G V R Q Q G I T I L P G
S S I S G A G L G H A A Y L S P G S G
A A V S P S H A S S S G D S P K A A K
I P P R P P P K P K K K M S V T T T R
S G Q G S T S Q L F D D E G E D G T E
V D P A F L Y K V V V P R A R D P P V
A T M V S K G E E L F T G V V P I L V
E L D G D V N G H K F S V S G E G E G
D A T Y G K L T L K F I C T T G K L P
V P W P T L V T T L T Y G V Q C F S R
Y P D H M K Q H D F F K S A M P E G Y
V Q E R T I F F K D D G N Y K T R A E
V K F E G D T L V N R I E L K G I D F
K E D G N I L G H K L E Y N Y N S H N
V Y I M A D K Q K N G I K V N F K I R
H N I E D G S V Q L A D H Y Q Q N T P
I G D G P V L L P D N H Y L S T Q S A
L S K D P N E K R D H M V L L E F V T
A A G I T L G M D E L Y K •
Good sequence for B1 & B4
IC1 modified oligo746 apK5_seq_int_3'
∆1 ConfirmedIC1
oligo240
∆IC2 ChloeL
4749
4806
4863
4920
4977
5034
5091
5148
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
1135
TCG CGC AAC CTG GCG CTC ATT GCC ACC ACC AAC AGC AGT CGC CAG CAC CAG CAC CAC
CAT CAG CTG CAG CAG CAG CAG CAG CAC CAC CAC CAC CAC CAG CAG CAA CAA CAA CAG
CAG CAG CAG CAG CAA CAT CCG CTG GCC ACC ACA TCG CCC TTC CTG CCC GCA CCC GTC
GTC TAT TCG CCG GCC ACG GGT GTG GTC ATG AAA CAG GGA TAT ATG ACC ATT CCG CGC
AAG CCG CGC GCT CCC AGC TGG GCG CCC AGT ACT TCC GGT GCC GCT GGC CAC GGA TCC
ATT CAG CTA AGT GAA TTC CAG AGC CCC ACA TCG CCG AAT CCC AGC GAG ACT GGC ACC
GCC ACC ACC GCG GAA CTG CAG GCG GAG CCA GTG TAC GAC AAC TTG GGA TTG CGA ACC
ACT GCC GGC GGC AAC TCC ACC CTC AAT CTG ACC AAG ATC GCC GGC TCA CAG GGG GGC
AGC GCG TTG GAC CGC GAG TAA CGG TGG TGG TTG TCG TCA GCG GTC GTG GTC GTG GTG
GTA GTC GAC GTC GTC GTC GTC GTC GTG GTG GTG GTG GTG GTC GTC GTT GTT GTT GTC
GTC GTC GTC GTC GTT GTA GGC GAC CGG TGG TGT AGC GGG AAG GAC GGG CGT GGG CAG
CAG ATA AGC GGC CGG TGC CCA CAC CAG TAC TTT GTC CCT ATA TAC TGG TAA GGC GCG
TTC GGC GCG CGA GGG TCG ACC CGC GGG TCA TGA AGG CCA CGG CGA CCG GTG CCT AGG
TAA GTC GAT TCA CTT AAG GTC TCG GGG TGT AGC GGC TTA GGG TCG CTC TGA CCG TGG
CGG TGG TGG CGC CTT GAC GTC CGC CTC GGT CAC ATG CTG TTG AAC CCT AAC GCT TGG
TGA CGG CCG CCG TTG AGG TGG GAG TTA GAC TGG TTC TAG CGG CCG AGT GTC CCC CCG
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P Y G I R
S P P S L T S P V Y T H M T P H G I Y
G T K T M T A P H N G F M T L Q H P K
S R N L A L I A T T N S S R Q H Q H H
H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V
V Y S P A T G V V M K Q G Y M T I P R
K P R A P S W A P S T S G A A G H G S
I Q L S E F Q S P T S P N P S E T G T
A T T A E L Q A E P V Y D N L G L R T
T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T
S V S S A T N A S K I Y E P I H E L I
Q Q Q Q Q L Q Q Q Q Q Q Q Q Q R L G S
M D T E P L Y G V R Q Q G I T I L P G
S S I S G A G L G H A A Y L S P G S G
A A V S P S H A S S S G D S P K A A K
I P P R P P P K P K K K M S V T T T R
S G Q G S T S Q L F D D E G E D G T E
V D P A F L Y K V V V P R A R D P P V
A T M V S K G E E L F T G V V P I L V
E L D G D V N G H K F S V S G E G E G
D A T Y G K L T L K F I C T T G K L P
V P W P T L V T T L T Y G V Q C F S R
Y P D H M K Q H D F F K S A M P E G Y
V Q E R T I F F K D D G N Y K T R A E
V K F E G D T L V N R I E L K G I D F
K E D G N I L G H K L E Y N Y N S H N
V Y I M A D K Q K N G I K V N F K I R
H N I E D G S V Q L A D H Y Q Q N T P
I G D G P V L L P D N H Y L S T Q S A
L S K D P N E K R D H M V L L E F V T
A A G I T L G M D E L Y K •
IC3 modified IC3
oligo241
∆45 Confirmed∆4 ConfirmedIC4 modified
IC4
5205
5262
5319
5376
5433
5490
5547
5604
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
1135
GCT GGT CAG CAG TAC TCG ATG CGG GAC CGA CCA CTT CCG GCC ACG CCC AGC CTG ACA
TCG GTG TCC TCG GCG ACC AAT GCC AGT AAG ATT TAC GAG CCC ATA CAC GAG CTG ATT
CAG CAG CAA CAG CAG TTG CAA CAA CAA CAA CAG CAG CAG CAG CAG CGA CTG GGC TCC
ATG GAC ACG GAA CCC CTG TAC GGA GTT CGG CAA CAG GGG ATC ACG ATA CTG CCC GGC
TCG AGC ATT AGC GGT GCC GGA CTG GGC CAC GCC GCC TAC CTT TCA CCC GGC TCG GGT
GCC GCC GTA TCG CCA AGC CAC GCC AGC AGC AGC GGT GAC TCT CCG AAG GCC GCC AAG
ATC CCA CCA CGC CCA CCA CCG AAG CCC AAG AAG AAG ATG TCC GTG ACG ACG ACG CGC
AGC GGC CAG GGC AGC ACC AGC CAG CTC TTC GAC GAC GAG GGC GAG GAT GGC ACC GAG
CGA CCA GTC GTC ATG AGC TAC GCC CTG GCT GGT GAA GGC CGG TGC GGG TCG GAC TGT
AGC CAC AGG AGC CGC TGG TTA CGG TCA TTC TAA ATG CTC GGG TAT GTG CTC GAC TAA
GTC GTC GTT GTC GTC AAC GTT GTT GTT GTT GTC GTC GTC GTC GTC GCT GAC CCG AGG
TAC CTG TGC CTT GGG GAC ATG CCT CAA GCC GTT GTC CCC TAG TGC TAT GAC GGG CCG
AGC TCG TAA TCG CCA CGG CCT GAC CCG GTG CGG CGG ATG GAA AGT GGG CCG AGC CCA
CGG CGG CAT AGC GGT TCG GTG CGG TCG TCG TCG CCA CTG AGA GGC TTC CGG CGG TTC
TAG GGT GGT GCG GGT GGT GGC TTC GGG TTC TTC TTC TAC AGG CAC TGC TGC TGC GCG
TCG CCG GTC CCG TCG TGG TCG GTC GAG AAG CTG CTG CTC CCG CTC CTA CCG TGG CTC
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P Y G I R
S P P S L T S P V Y T H M T P H G I Y
G T K T M T A P H N G F M T L Q H P K
S R N L A L I A T T N S S R Q H Q H H
H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V
V Y S P A T G V V M K Q G Y M T I P R
K P R A P S W A P S T S G A A G H G S
I Q L S E F Q S P T S P N P S E T G T
A T T A E L Q A E P V Y D N L G L R T
T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T
S V S S A T N A S K I Y E P I H E L I
Q Q Q Q Q L Q Q Q Q Q Q Q Q Q R L G S
M D T E P L Y G V R Q Q G I T I L P G
S S I S G A G L G H A A Y L S P G S G
A A V S P S H A S S S G D S P K A A K
I P P R P P P K P K K K M S V T T T R
S G Q G S T S Q L F D D E G E D G T E
V D P A F L Y K V V V P R A R D P P V
A T M V S K G E E L F T G V V P I L V
E L D G D V N G H K F S V S G E G E G
D A T Y G K L T L K F I C T T G K L P
V P W P T L V T T L T Y G V Q C F S R
Y P D H M K Q H D F F K S A M P E G Y
V Q E R T I F F K D D G N Y K T R A E
V K F E G D T L V N R I E L K G I D F
K E D G N I L G H K L E Y N Y N S H N
V Y I M A D K Q K N G I K V N F K I R
H N I E D G S V Q L A D H Y Q Q N T P
I G D G P V L L P D N H Y L S T Q S A
L S K D P N E K R D H M V L L E F V T
A A G I T L G M D E L Y K •
End of Good sequence for B1 & B4
∆1235 Confirmed∆5 ConfirmedIC5
good sequence 5' to this siteIC6 (PDZ)
5661
5718
5775
5832
5889
5946
6003
6060
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
1135
GTC GAC CCA GCT TTC TTG TAC AAA GTG GTG GTA CCG CGG GCC CGG GAT CCA CCG GTC
GCC ACC ATG GTG AGC AAG GGC GAG GAG CTG TTC ACC GGG GTG GTG CCC ATC CTG GTC
GAG CTG GAC GGC GAC GTA AAC GGC CAC AAG TTC AGC GTG TCC GGC GAG GGC GAG GGC
GAT GCC ACC TAC GGC AAG CTG ACC CTG AAG TTC ATC TGC ACC ACC GGC AAG CTG CCC
GTG CCC TGG CCC ACC CTC GTG ACC ACC CTG ACC TAC GGC GTG CAG TGC TTC AGC CGC
TAC CCC GAC CAC ATG AAG CAG CAC GAC TTC TTC AAG TCC GCC ATG CCC GAA GGC TAC
GTC CAG GAG CGC ACC ATC TTC TTC AAG GAC GAC GGC AAC TAC AAG ACC CGC GCC GAG
GTG AAG TTC GAG GGC GAC ACC CTG GTG AAC CGC ATC GAG CTG AAG GGC ATC GAC TTC
CAG CTG GGT CGA AAG AAC ATG TTT CAC CAC CAT GGC GCC CGG GCC CTA GGT GGC CAG
CGG TGG TAC CAC TCG TTC CCG CTC CTC GAC AAG TGG CCC CAC CAC GGG TAG GAC CAG
CTC GAC CTG CCG CTG CAT TTG CCG GTG TTC AAG TCG CAC AGG CCG CTC CCG CTC CCG
CTA CGG TGG ATG CCG TTC GAC TGG GAC TTC AAG TAG ACG TGG TGG CCG TTC GAC GGG
CAC GGG ACC GGG TGG GAG CAC TGG TGG GAC TGG ATG CCG CAC GTC ACG AAG TCG GCG
ATG GGG CTG GTG TAC TTC GTC GTG CTG AAG AAG TTC AGG CGG TAC GGG CTT CCG ATG
CAG GTC CTC GCG TGG TAG AAG AAG TTC CTG CTG CCG TTG ATG TTC TGG GCG CGG CTC
CAC TTC AAG CTC CCG CTG TGG GAC CAC TTG GCG TAG CTC GAC TTC CCG TAG CTG AAG
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P Y G I R
S P P S L T S P V Y T H M T P H G I Y
G T K T M T A P H N G F M T L Q H P K
S R N L A L I A T T N S S R Q H Q H H
H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V
V Y S P A T G V V M K Q G Y M T I P R
K P R A P S W A P S T S G A A G H G S
I Q L S E F Q S P T S P N P S E T G T
A T T A E L Q A E P V Y D N L G L R T
T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T
S V S S A T N A S K I Y E P I H E L I
Q Q Q Q Q L Q Q Q Q Q Q Q Q Q R L G S
M D T E P L Y G V R Q Q G I T I L P G
S S I S G A G L G H A A Y L S P G S G
A A V S P S H A S S S G D S P K A A K
I P P R P P P K P K K K M S V T T T R
S G Q G S T S Q L F D D E G E D G T E
V D P A F L Y K V V V P R A R D P P V
A T M V S K G E E L F T G V V P I L V
E L D G D V N G H K F S V S G E G E G
D A T Y G K L T L K F I C T T G K L P
V P W P T L V T T L T Y G V Q C F S R
Y P D H M K Q H D F F K S A M P E G Y
V Q E R T I F F K D D G N Y K T R A E
V K F E G D T L V N R I E L K G I D F
K E D G N I L G H K L E Y N Y N S H N
V Y I M A D K Q K N G I K V N F K I R
H N I E D G S V Q L A D H Y Q Q N T P
I G D G P V L L P D N H Y L S T Q S A
L S K D P N E K R D H M V L L E F V T
A A G I T L G M D E L Y K •
attL2
attB2KpnI oligo 445 T7
EGFPN1 polyl inker
EGFPN1
#54
6117
6174
6231
6288
6345
6402
6459
6516
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
1135
AAG GAG GAC GGC AAC ATC CTG GGG CAC AAG CTG GAG TAC AAC TAC AAC AGC CAC AAC
GTC TAT ATC ATG GCC GAC AAG CAG AAG AAC GGC ATC AAG GTG AAC TTC AAG ATC CGC
CAC AAC ATC GAG GAC GGC AGC GTG CAG CTC GCC GAC CAC TAC CAG CAG AAC ACC CCC
ATC GGC GAC GGC CCC GTG CTG CTG CCC GAC AAC CAC TAC CTG AGC ACC CAG TCC GCC
CTG AGC AAA GAC CCC AAC GAG AAG CGC GAT CAC ATG GTC CTG CTG GAG TTC GTG ACC
GCC GCC GGG ATC ACT CTC GGC ATG GAC GAG CTG TAC AAG TAA AGCGGCCGCGACTCTAGAGGAT
CTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAA
TATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTGTGTATTTTAGATTCCAACCTATGGAAC
TGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGA
TGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTC
AGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGA
TTC CTC CTG CCG TTG TAG GAC CCC GTG TTC GAC CTC ATG TTG ATG TTG TCG GTG TTG
CAG ATA TAG TAC CGG CTG TTC GTC TTC TTG CCG TAG TTC CAC TTG AAG TTC TAG GCG
GTG TTG TAG CTC CTG CCG TCG CAC GTC GAG CGG CTG GTG ATG GTC GTC TTG TGG GGG
TAG CCG CTG CCG GGG CAC GAC GAC GGG CTG TTG GTG ATG GAC TCG TGG GTC AGG CGG
GAC TCG TTT CTG GGG TTG CTC TTC GCG CTA GTG TAC CAG GAC GAC CTC AAG CAC TGG
CGG CGG CCC TAG TGA GAG CCG TAC CTG CTC GAC ATG TTC ATT TCGCCGGCGCTGAGATCTCCTA
GAAACACTTCCTTGGAATGAAGACACCACACTGTATTAACCTGTTTGATGGATGTCTCTAAATTTCGAGATTCCATTT
ATATTTTAAAAATTCACATATTACACAATTTGATGACTAAGATTAACAAACACATAAAATCTAAGGTTGGATACCTTG
ACTACTTACCCTCGTCACCACCTTACGGAAATTACTCCTTTTGGACAAAACGAGTCTTCTTTACGGTAGATCACTACT
ACTCCGATGACGACTGAGAGTTGTAAGATGAGGAGGTTTTTTCTTCTCTTTCCATCTTCTGGGGTTCCTGAAAGGAAG
TCTTAACGATTCAAAAAACTCAGTACGACACAAATCATTATCTTGAGAACGAACGAAACGATAAATGTGGTGTTTCCT
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N V I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P Y G I R
S P P S L T S P V Y T H M T P H G I Y
G T K T M T A P H N G F M T L Q H P K
S R N L A L I A T T N S S R Q H Q H H
H Q L Q Q Q Q Q H H H H H Q Q Q Q Q Q
Q Q Q Q Q H P L A T T S P F L P A P V
V Y S P A T G V V M K Q G Y M T I P R
K P R A P S W A P S T S G A A G H G S
I Q L S E F Q S P T S P N P S E T G T
A T T A E L Q A E P V Y D N L G L R T
T A G G N S T L N L T K I A G S Q G G
A G Q Q Y S M R D R P L P A T P S L T
S V S S A T N A S K I Y E P I H E L I
Q Q Q Q Q L Q Q Q Q Q Q Q Q Q R L G S
M D T E P L Y G V R Q Q G I T I L P G
S S I S G A G L G H A A Y L S P G S G
A A V S P S H A S S S G D S P K A A K
I P P R P P P K P K K K M S V T T T R
S G Q G S T S Q L F D D E G E D G T E
V D P A F L Y K V V V P R A R D P P V
A T M V S K G E E L F T G V V P I L V
E L D G D V N G H K F S V S G E G E G
D A T Y G K L T L K F I C T T G K L P
V P W P T L V T T L T Y G V Q C F S R
Y P D H M K Q H D F F K S A M P E G Y
V Q E R T I F F K D D G N Y K T R A E
V K F E G D T L V N R I E L K G I D F
K E D G N I L G H K L E Y N Y N S H N
V Y I M A D K Q K N G I K V N F K I R
H N I E D G S V Q L A D H Y Q Q N T P
I G D G P V L L P D N H Y L S T Q S A
L S K D P N E K R D H M V L L E F V T
A A G I T L G M D E L Y K •
Xba I SV40 Poly A
6573
6630
6687
6744
6801
6858
6922
7000
7078
7156
7234
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
1135
AAAAGCTGCACTGCTATACAAGAAAATTATGGAAAAATATTCTGTAACCTTTATAAGTAGGCATAACAGTTATAATCA
TAACATACTGTTTTTTCTTACTCCACACAGGCATAGAGTGTCTGCTATTAATAACTATGCTCAAAAATTGTGTACCTT
TAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATATTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATA
CCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAA
TTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAG
CATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCGGATCCACTA
GAAGGCCTTAGTATGTATGTAAGTTAATAAAACCCTTTTTTGGAGAATGTAGATTTAAAAAAACATATTTTTTTTTTA
TTTTTTACTGCACTGGACATCATTGAACTTATCTGATCAGTTTTAAATTTACTTCGATCCAAGGGTATTTGAAGTACC
AGGTTCTTTCGATTACCTCTCACTCAAAATGACATTCCACTCAAAGTCAGCGCTGTTTGCCTCCTTCTCTGTCCACAG
AAATATCGCCGTCTCTTTCGCCGCTGCGTCCGCTATCTCTTTCGCCACCGTTTGTAGCGTTACCTAGCGTCAATGTCC
GCCTTCAGTTGCACTTTGTCAGCGGTTTCGTGACGAAGCTCCAAGCGGTTTACGCCATCAATTAAACACAAAGTGCTG
TGCCAAAACTCCTCTCGCTTCTTATTTTTGTTTGTTTTTTGAGTGATTGGGGTGGTGATTGGTTTTGGGTGGGTAAGC
AGGGGAAAGTGTGAAAAATCCCGGCAATGGGCCAAGAGGATCAGGAGCTATTAATTCGCGGAGGCAGCAAACACCCAT
CTGCCGAGCATCTGAACAATGTGAGTAGTACATGTGCATACATCTTAAGTTCACTTGATCTATAGGAACTGCGATTGC
AACATCAAATTGTCTGCGGCGTGAGAACTGCGACCCACAAAAATCCCAAACCGCAATCGCACAAACAAATAGTGACAC
GAAACAGATTATTCTGGTAGCTGTGCTCGCTATATAAGACAATTTTTAAGATCATATCATGATCAAGACATCTAAAGG
TTTTCGACGTGACGATATGTTCTTTTAATACCTTTTTATAAGACATTGGAAATATTCATCCGTATTGTCAATATTAGT
ATTGTATGACAAAAAAGAATGAGGTGTGTCCGTATCTCACAGACGATAATTATTGATACGAGTTTTTAACACATGGAA
ATCGAAAAATTAAACATTTCCCCAATTATTCCTTATAAACTACATATCACGGAACTGATCTCTAGTATTAGTCGGTAT
GGTGTAAACATCTCCAAAATGAACGAAATTTTTTGGAGGGTGTGGAGGGGGACTTGGACTTTGTATTTTACTTACGTT
AACAACAACAATTGAACAAATAACGTCGAATATTACCAATGTTTATTTCGTTATCGTAGTGTTTAAAGTGTTTATTTC
GTAAAAAAAGTGACGTAAGATCAACACCAAACAGGTTTGAGTAGTTACATAGAATAGTACAGACCTAGCCTAGGTGAT
CTTCCGGAATCATACATACATTCAATTATTTTGGGAAAAAACCTCTTACATCTAAATTTTTTTGTATAAAAAAAAAAT
AAAAAATGACGTGACCTGTAGTAACTTGAATAGACTAGTCAAAATTTAAATGAAGCTAGGTTCCCATAAACTTCATGG
TCCAAGAAAGCTAATGGAGAGTGAGTTTTACTGTAAGGTGAGTTTCAGTCGCGACAAACGGAGGAAGAGACAGGTGTC
TTTATAGCGGCAGAGAAAGCGGCGACGCAGGCGATAGAGAAAGCGGTGGCAAACATCGCAATGGATCGCAGTTACAGG
CGGAAGTCAACGTGAAACAGTCGCCAAAGCACTGCTTCGAGGTTCGCCAAATGCGGTAGTTAATTTGTGTTTCACGAC
ACGGTTTTGAGGAGAGCGAAGAATAAAAACAAACAAAAAACTCACTAACCCCACCACTAACCAAAACCCACCCATTCG
TCCCCTTTCACACTTTTTAGGGCCGTTACCCGGTTCTCCTAGTCCTCGATAATTAAGCGCCTCCGTCGTTTGTGGGTA
GACGGCTCGTAGACTTGTTACACTCATCATGTACACGTATGTAGAATTCAAGTGAACTAGATATCCTTGACGCTAACG
TTGTAGTTTAACAGACGCCGCACTCTTGACGCTGGGTGTTTTTAGGGTTTGGCGTTAGCGTGTTTGTTTATCACTGTG
CTTTGTCTAATAAGACCATCGACACGAGCGATATATTCTGTTAAAAATTCTAGTATAGTACTAGTTCTGTAGATTTCC
HpaI
B a m H I
white gene
7312
7390
7468
7546
7624
7702
7780
7858
7936
8014
8092
8170
8248
8326
8404
8482
CATTCATTTTCGACTACATTCTTTTTTACAAAAAATATAACAACCAGATATTTTAAGCTGATCCTAGATGCACAAAAA
ATAAATAAAAGTATAAACCTACTTCGTAGGATACTTCGTTTTGTTCGGGGTTAGATGAGCATAACGCTTGTAGTTGAT
ATTTGAGATCCCCTATCATTGCAGGGTGACAGCGGAGCGGCTTCGCAGAGCTGCATTAACCAGGGCTTCGGGCAGGCC
AAAAACTACGGCACGCTCCTGCCACCCAGTCCGCCGGAGGACTCCGGTTCAGGGAGCGGCCAACTAGCCGAGAACCTC
ACCTATGCCTGGCACAATATGGACATCTTTGGGGCGGTCAATCAGCCGGGCTCCGGATGGCGGCAGCTGGTCAACCGG
ACACGCGGACTATTCTGCAACGAGCGACACATACCGGCGCCCAGGAAACATTTGCTCAAGAACGGTGAGTTTCTATTC
GCAGTCGGCTGATCTGTGTGAAATCTTAATAAAGGGTCCAATTACCAATTTGAAACTCAGTTTGCGGCGTGGCCTATC
CGGGCGAACTTTTGGCCGTGATGGGCAGTTCCGGTGCCGGAAAGACGACCCTGCTGAATGCCCTTGCCTTTCGATCGC
CGCAGGGCATCCAAGTATCGCCATCCGGGATGCGACTGCTCAATGGCCAACCTGTGGACGCCAAGGAGATGCAGGCCA
GGTGCGCCTATGTCCAGCAGGATGACCTCTTTATCGGCTCCCTAACGGCCAGGGAACACCTGATTTTCCAGGCCATGG
TGCGGATGCCACGACATCTGACCTATCGGCAGCGAGTGGCCCGCGTGGATCAGGTGATCCAGGAGCTTTCGCTCAGCA
AATGTCAGCACACGATCATCGGTGTGCCCGGCAGGGTGAAAGGTCTGTCCGGCGGAGAAAGGAAGCGTCTGGCATTCG
CCTCCGAGGCACTAACCGATCCGCCGCTTCTGATCTGCGATGAGCCCACCTCCGGACTGGACTCATTTACCGCCCACA
GCGTCGTCCAGGTGCTGAAGAAGCTGTCGCAGAAGGGCAAGACCGTCATCCTGACCATTCATCAGCCGTCTTCCGAGC
TGTTTGAGCTCTTTGACAAGATCCTTCTGATGGCCGAGGGCAGGGTAGCTTTCTTGGGCACTCCCAGCGAAGCCGTCG
ACTTCTTTTCCTAGTGAGTTCGATGTGTTTATTAAGGGTATCTAGCATTACATTACATCTCAACTCCTATCCAGCGTG
GGTGCCCAGTGTCCTACCAACTACAATCCGGCGGACTTTTACGTACAGGTGTTGGCCGTTGTGCCCGGACGGGAGATC
GTAAGTAAAAGCTGATGTAAGAAAAAATGTTTTTTATATTGTTGGTCTATAAAATTCGACTAGGATCTACGTGTTTTT
TATTTATTTTCATATTTGGATGAAGCATCCTATGAAGCAAAACAAGCCCCAATCTACTCGTATTGCGAACATCAACTA
TAAACTCTAGGGGATAGTAACGTCCCACTGTCGCCTCGCCGAAGCGTCTCGACGTAATTGGTCCCGAAGCCCGTCCGG
TTTTTGATGCCGTGCGAGGACGGTGGGTCAGGCGGCCTCCTGAGGCCAAGTCCCTCGCCGGTTGATCGGCTCTTGGAG
TGGATACGGACCGTGTTATACCTGTAGAAACCCCGCCAGTTAGTCGGCCCGAGGCCTACCGCCGTCGACCAGTTGGCC
TGTGCGCCTGATAAGACGTTGCTCGCTGTGTATGGCCGCGGGTCCTTTGTAAACGAGTTCTTGCCACTCAAAGATAAG
CGTCAGCCGACTAGACACACTTTAGAATTATTTCCCAGGTTAATGGTTAAACTTTGAGTCAAACGCCGCACCGGATAG
GCCCGCTTGAAAACCGGCACTACCCGTCAAGGCCACGGCCTTTCTGCTGGGACGACTTACGGGAACGGAAAGCTAGCG
GCGTCCCGTAGGTTCATAGCGGTAGGCCCTACGCTGACGAGTTACCGGTTGGACACCTGCGGTTCCTCTACGTCCGGT
CCACGCGGATACAGGTCGTCCTACTGGAGAAATAGCCGAGGGATTGCCGGTCCCTTGTGGACTAAAAGGTCCGGTACC
ACGCCTACGGTGCTGTAGACTGGATAGCCGTCGCTCACCGGGCGCACCTAGTCCACTAGGTCCTCGAAAGCGAGTCGT
TTACAGTCGTGTGCTAGTAGCCACACGGGCCGTCCCACTTTCCAGACAGGCCGCCTCTTTCCTTCGCAGACCGTAAGC
GGAGGCTCCGTGATTGGCTAGGCGGCGAAGACTAGACGCTACTCGGGTGGAGGCCTGACCTGAGTAAATGGCGGGTGT
CGCAGCAGGTCCACGACTTCTTCGACAGCGTCTTCCCGTTCTGGCAGTAGGACTGGTAAGTAGTCGGCAGAAGGCTCG
ACAAACTCGAGAAACTGTTCTAGGAAGACTACCGGCTCCCGTCCCATCGAAAGAACCCGTGAGGGTCGCTTCGGCAGC
TGAAGAAAAGGATCACTCAAGCTACACAAATAATTCCCATAGATCGTAATGTAATGTAGAGTTGAGGATAGGTCGCAC
CCACGGGTCACAGGATGGTTGATGTTAGGCCGCCTGAAAATGCATGTCCACAACCGGCAACACGGGCCTGCCCTCTAG
8560
8638
8716
8794
8872
8950
9028
9106
9184
9262
9340
9418
9496
9574
9652
9730
9808
GAGTCCCGTGATCGGATCGCCAAGATATGCGACAATTTTGCTATTAGCAAAGTAGCCCGGGATATGGAGCAGTTGTTG
GCCACCAAAAATTTGGAGAAGCCACTGGAGCAGCCGGAGAATGGGTACACCTACAAGGCCACCTGGTTCATGCAGTTC
CGGGCGGTCCTGTGGCGATCCTGGCTGTCGGTGCTCAAGGAACCACTCCTCGTAAAAGTGCGACTTATTCAGACAACG
GTGAGTGGTTCCAGTGGAAACAAATGATATAACGCTTACAATTCTTGGAAACAAATTCGCTAGATTTTAGTTAGAATT
GCCTGATTCCACACCCTTCTTAGTTTTTTTCAATGAGATGTATAGTTTATAGTTTTGCAGAAAATAAATAAATTTCAT
TTAACTCGCGAACATGTTGAAGATATGAATATTAATGAGATGCGAGTAACATTTTAATTTGCAGATGGTTGCCATCTT
GATTGGCCTCATCTTTTTGGGCCAACAACTCACGCAAGTGGGCGTGATGAATATCAACGGAGCCATCTTCCTCTTCCT
GACCAACATGACCTTTCAAAACGTCTTTGCCACGATAAATGTAAGTCTTGTTTAGAATACATTTGCATATTAATAATT
TACTAACTTTCTAATGAATCGATTCGATTTAGGTGTTCACCTCAGAGCTGCCAGTTTTTATGAGGGAGGCCCGAAGTC
GACTTTATCGCTGTGACACATACTTTCTGGGCAAAACGATTGCCGAATTACCGCTTTTTCTCACAGTGCCACTGGTCT
TCACGGCGATTGCCTATCCGATGATCGGACTGCGGGCCGGAGTGCTGCACTTCTTCAACTGCCTGGCGCTGGTCACTC
TGGTGGCCAATGTGTCAACGTCCTTCGGATATCTAATATCCTGCGCCAGCTCCTCGACCTCGATGGCGCTGTCTGTGG
GTCCGCCGGTTATCATACCATTCCTGCTCTTTGGCGGCTTCTTCTTGAACTCGGGCTCGGTGCCAGTATACCTCAAAT
GGTTGTCGTACCTCTCATGGTTCCGTTACGCCAACGAGGGTCTGCTGATTAACCAATGGGCGGACGTGGAGCCGGGCG
AAATTAGCTGCACATCGTCGAACACCACGTGCCCCAGTTCGGGCAAGGTCATCCTGGAGACGCTTAACTTCTCCGCCG
CCGATCTGCCGCTGGACTACGTGGGTCTGGCCATTCTCATCGTGAGCTTCCGGGTGCTCGCATATCTGGCTCTAAGAC
TTCGGGCCCGACGCAAGGAGTAGCCGACATATATCCGAAATAACTGCTTGTTTTTTTTTTTACCATTATTACCATCGT
CTCAGGGCACTAGCCTAGCGGTTCTATACGCTGTTAAAACGATAATCGTTTCATCGGGCCCTATACCTCGTCAACAAC
CGGTGGTTTTTAAACCTCTTCGGTGACCTCGTCGGCCTCTTACCCATGTGGATGTTCCGGTGGACCAAGTACGTCAAG
GCCCGCCAGGACACCGCTAGGACCGACAGCCACGAGTTCCTTGGTGAGGAGCATTTTCACGCTGAATAAGTCTGTTGC
CACTCACCAAGGTCACCTTTGTTTACTATATTGCGAATGTTAAGAACCTTTGTTTAAGCGATCTAAAATCAATCTTAA
CGGACTAAGGTGTGGGAAGAATCAAAAAAAGTTACTCTACATATCAAATATCAAAACGTCTTTTATTTATTTAAAGTA
AATTGAGCGCTTGTACAACTTCTATACTTATAATTACTCTACGCTCATTGTAAAATTAAACGTCTACCAACGGTAGAA
CTAACCGGAGTAGAAAAACCCGGTTGTTGAGTGCGTTCACCCGCACTACTTATAGTTGCCTCGGTAGAAGGAGAAGGA
CTGGTTGTACTGGAAAGTTTTGCAGAAACGGTGCTATTTACATTCAGAACAAATCTTATGTAAACGTATAATTATTAA
ATGATTGAAAGATTACTTAGCTAAGCTAAATCCACAAGTGGAGTCTCGACGGTCAAAAATACTCCCTCCGGGCTTCAG
CTGAAATAGCGACACTGTGTATGAAAGACCCGTTTTGCTAACGGCTTAATGGCGAAAAAGAGTGTCACGGTGACCAGA
AGTGCCGCTAACGGATAGGCTACTAGCCTGACGCCCGGCCTCACGACGTGAAGAAGTTGACGGACCGCGACCAGTGAG
ACCACCGGTTACACAGTTGCAGGAAGCCTATAGATTATAGGACGCGGTCGAGGAGCTGGAGCTACCGCGACAGACACC
CAGGCGGCCAATAGTATGGTAAGGACGAGAAACCGCCGAAGAAGAACTTGAGCCCGAGCCACGGTCATATGGAGTTTA
CCAACAGCATGGAGAGTACCAAGGCAATGCGGTTGCTCCCAGACGACTAATTGGTTACCCGCCTGCACCTCGGCCCGC
TTTAATCGACGTGTAGCAGCTTGTGGTGCACGGGGTCAAGCCCGTTCCAGTAGGACCTCTGCGAATTGAAGAGGCGGC
GGCTAGACGGCGACCTGATGCACCCAGACCGGTAAGAGTAGCACTCGAAGGCCCACGAGCGTATAGACCGAGATTCTG
AAGCCCGGGCTGCGTTCCTCATCGGCTGTATATAGGCTTTATTGACGAACAAAAAAAAAAATGGTAATAATGGTAGCA
9886
9964
10042
10120
10198
10276
10354
10432
10510
10588
10666
10744
10822
10900
10978
11056
11134
GTTTACTGTTTATTGCCCCCTCAAAAAGCTAATGTAATTATATTTGTGCCAATAAAAACAAGATATGACCTATAGAAT
ACAAGTATTTCCCCTTCGAACATCCCCACAAGTAGACTTTGGATTTGTCTTCTAACCAAAAGACTTACACACCTGCAT
ACCTTACATCAAAAACTCGTTTATCGCTACATAAAACACCGGGATATATTTTTTATATACATACTTTTCAAATCGCGC
GCCCTCTTCATAATTCACCTCCACCACACCACGTTTCGTAGTTGCTCTTTCGCTGTCTCCCACCCGCTCTCCGCAACA
CATTCACCTTTTGTTCGACGACCTTGGAGCGACTGTCGTTAGTTCCGCGCGATTCGGTTCGCTCAAATGGTTCCGAGT
GGTTCATTTCGTCTCAATAGAAATTAGTAATAAATATTTGTATGTACAATTTATTTGCTCCAATATATTTGTATATAT
TTCCCTCACAGCTATATTTATTCTAATTTAATATTATGACTTTTTAAGGTAATTTTTTGTGACCTGTTCGGAGTGATT
AGCGTTACAATTTGAACTGAAAGTGACATCCAGTGTTTGTTCCTTGTGTAGATGCATCTCAAAAAAATGGTGGGCATA
ATAGTGTTGTTTATATATATCAAAAATAACAACTATAATAATAAGAATACATTTAATTTAGAAAATGCTTGGATTTCA
CTGGAACTAGAATTAATTCGGCTGCTGCTCTAAACGACGCATTTCGTACTCCAAAGTACGAATTTTTTCCCTCAAGCT
CTTATTTTCATTAAACAATGAACAGGACCTAACGCACAGTCACGTTATTGTTTACATAAATGATTTTTTTTACTATTC
AAACTTACTCTGTTTGTGTACTCCCACTGGTATAGCCTTCTTTTATCTTTTCTGGTTCAGGCTCTATCACTTTACTAG
GTACGGCATCTGCGTTGAGTCGCCTCCTTTTAAATGTCTGACCTTTTGCAGGTGCAGCCTTCCACTGCGAATCATTAA
AGTGGGTATCACAAATTTGGGAGTTTTCACCAAGGCTGCACCCAAGGCTCTGCTCCCACAATTTTCTCTTAATAGCAC
ACTTCGGCACGTGAATTAATTTTACTCCAGTCACAGCTTTGCAGCAAAATTTGCAATATTTCATTTTTTTTTATTCCA
CGTAAGGGTTAATGTTTTCAAAAAAAAATTCGTCCGCACACAACCTTTCCTCTCAACAAGCAAACGTGCACTGAATTT
CAAATGACAAATAACGGGGGAGTTTTTCGATTACATTAATATAAACACGGTTATTTTTGTTCTATACTGGATATCTTA
TGTTCATAAAGGGGAAGCTTGTAGGGGTGTTCATCTGAAACCTAAACAGAAGATTGGTTTTCTGAATGTGTGGACGTA
TGGAATGTAGTTTTTGAGCAAATAGCGATGTATTTTGTGGCCCTATATAAAAAATATATGTATGAAAAGTTTAGCGCG
CGGGAGAAGTATTAAGTGGAGGTGGTGTGGTGCAAAGCATCAACGAGAAAGCGACAGAGGGTGGGCGAGAGGCGTTGT
GTAAGTGGAAAACAAGCTGCTGGAACCTCGCTGACAGCAATCAAGGCGCGCTAAGCCAAGCGAGTTTACCAAGGCTCA
CCAAGTAAAGCAGAGTTATCTTTAATCATTATTTATAAACATACATGTTAAATAAACGAGGTTATATAAACATATATA
AAGGGAGTGTCGATATAAATAAGATTAAATTATAATACTGAAAAATTCCATTAAAAAACACTGGACAAGCCTCACTAA
TCGCAATGTTAAACTTGACTTTCACTGTAGGTCACAAACAAGGAACACATCTACGTAGAGTTTTTTTACCACCCGTAT
TATCACAACAAATATATATAGTTTTTATTGTTGATATTATTATTCTTATGTAAATTAAATCTTTTACGAACCTAAAGT
GACCTTGATCTTAATTAAGCCGACGACGAGATTTGCTGCGTAAAGCATGAGGTTTCATGCTTAAAAAAGGGAGTTCGA
GAATAAAAGTAATTTGTTACTTGTCCTGGATTGCGTGTCAGTGCAATAACAAATGTATTTACTAAAAAAAATGATAAG
TTTGAATGAGACAAACACATGAGGGTGACCATATCGGAAGAAAATAGAAAAGACCAAGTCCGAGATAGTGAAATGATC
CATGCCGTAGACGCAACTCAGCGGAGGAAAATTTACAGACTGGAAAACGTCCACGTCGGAAGGTGACGCTTAGTAATT
TCACCCATAGTGTTTAAACCCTCAAAAGTGGTTCCGACGTGGGTTCCGAGACGAGGGTGTTAAAAGAGAATTATCGTG
TGAAGCCGTGCACTTAATTAAAATGAGGTCAGTGTCGAAACGTCGTTTTAAACGTTATAAAGTAAAAAAAAATAAGGT
GCATTCCCAATTACAAAAGTTTTTTTTTAAGCAGGCGTGTGTTGGAAAGGAGAGTTGTTCGTTTGCACGTGACTTAAA
11212
11290
11368
11446
11524
11602
11680
11758
11836
11914
11992
12070
12148
12226
12304
12382
AAGTGTATACTTCGGTAAGCTTCGGCTATCGACGGGACCACCTTATGTTATTTCATCATGTTCACATATGAAGCCATTCGAAGCCGATAGCTGCCCTGGTGGAATACAATAAAGTAGTAC
5' P
5' P
12460
GGCCAGACCCACGTAGTCCAGCGGCAGATCGGCGGCGGAGAAGTTAAGCGTCTCCAGGATGACCTTGCCCGAACTGGG
GCACGTGGTGTTCGACGATGTGCAGCTAATTTCGCCCGGCTCCACGTCCGCCCATTGGTTAATCAGCAGACCCTCGTT
GGCGTAACGGAACCATGAGAGGTACGACAACCATTTGAGGTATACTGGCACCGAGCCCGAGTTCAAGAAGAAGGCGTT
TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT
ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCT
GTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGT
TCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGA
GTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG
CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT
GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT
TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC
TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAA
TTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGA
GGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACG
GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAAT
AAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTG
CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC
ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTG
CAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT
GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTC
ATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAG
AACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG
TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAAC
AGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATA
CCGGTCTGGGTGCATCAGGTCGCCGTCTAGCCGCCGCCTCTTCAATTCGCAGAGGTCCTACTGGAACGGGCTTGACCC
CGTGCACCACAAGCTGCTACACGTCGATTAAAGCGGGCCGAGGTGCAGGCGGGTAACCAATTAGTCGTCTGGGAGCAA
CCGCATTGCCTTGGTACTCTCCATGCTGTTGGTAAACTCCATATGACCGTGGCTCGGGCTCAAGTTCTTCTTCCGCAA
AAAGGTATCCGAGGCGGGGGGACTGCTCGTAGTGTTTTTAGCTGCGAGTTCAGTCTCCACCGCTTTGGGCTGTCCTGA
TATTTCTATGGTCCGCAAAGGGGGACCTTCGAGGGAGCACGCGAGAGGACAAGGCTGGGACGGCGAATGGCCTATGGA
CAGGCGGAAAGAGGGAAGCCCTTCGCACCGCGAAAGAGTTACGAGTGCGACATCCATAGAGTCAAGCCACATCCAGCA
AGCGAGGTTCGACCCGACACACGTGCTTGGGGGGCAAGTCGGGCTGGCGACGCGGAATAGGCCATTGATAGCAGAACT
CAGGTTGGGCCATTCTGTGCTGAATAGCGGTGACCGTCGTCGGTGACCATTGTCCTAATCGTCTCGCTCCATACATCC
GCCACGATGTCTCAAGAACTTCACCACCGGATTGATGCCGATGTGATCTTCCTGTCATAAACCATAGACGCGAGACGA
CTTCGGTCAATGGAAGCCTTTTTCTCAACCATCGAGAACTAGGCCGTTTGTTTGGTGGCGACCATCGCCACCAAAAAA
ACAAACGTTCGTCGTCTAATGCGCGTCTTTTTTTCCTAGAGTTCTTCTAGGAAACTAGAAAAGATGCCCCAGACTGCG
AGTCACCTTGCTTTTGAGTGCAATTCCCTAAAACCAGTACTCTAATAGTTTTTCCTAGAAGTGGATCTAGGAAAATTT
AATTTTTACTTCAAAATTTAGTTAGATTTCATATATACTCATTTGAACCAGACTGTCAATGGTTACGAATTAGTCACT
CCGTGGATAGAGTCGCTAGACAGATAAAGCAAGTAGGTATCAACGGACTGAGGGGCAGCACATCTATTGATGCTATGC
CCTCCCGAATGGTAGACCGGGGTCACGACGTTACTATGGCGCTCTGGGTGCGAGTGGCCGAGGTCTAAATAGTCGTTA
TTTGGTCGGTCGGCCTTCCCGGCTCGCGTCTTCACCAGGACGTTGAAATAGGCGGAGGTAGGTCAGATAATTAACAAC
GGCCCTTCGATCTCATTCATCAAGCGGTCAATTATCAAACGCGTTGCAACAACGGTAACGATGTCCGTAGCACCACAG
TGCGAGCAGCAAACCATACCGAAGTAAGTCGAGGCCAAGGGTTGCTAGTTCCGCTCAATGTACTAGGGGGTACAACAC
GTTTTTTCGCCAATCGAGGAAGCCAGGAGGCTAGCAACAGTCTTCATTCAACCGGCGTCACAATAGTGAGTACCAATA
CCGTCGTGACGTATTAAGAGAATGACAGTACGGTAGGCATTCTACGAAAAGACACTGACCACTCATGAGTTGGTTCAG
TAAGACTCTTATCACATACGCCGCTGGCTCAACGAGAACGGGCCGCAGTTGTGCCCTATTATGGCGCGGTGTATCGTC
TTGAAATTTTCACGAGTAGTAACCTTTTGCAAGAAGCCCCGCTTTTGAGAGTTCCTAGAATGGCGACAACTCTAGGTC
AAGCTACATTGGGTGAGCACGTGGGTTGACTAGAAGTCGTAGAAAATGAAAGTGGTCGCAAAGACCCACTCGTTTTTG
TCCTTCCGTTTTACGGCGTTTTTTCCCTTATTCCCGCTGTGCCTTTACAACTTATGAGTATGAGAAGGAAAAAGTTAT
1
79
157
235
313
391
469
547
625
703
781
859
937
1015
1093
1171
1249
1327
1405
1483
1561
1639
1717
1795
TTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGG
GGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAA
TAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGA
GACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTG
TCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACCG
AATCGCGCGGAACTAACGACAGTCGCTCCAAGGTCGTCGAACAAAAGGTGAATGTGTTGCGGAGAGCGGGTGGGAGAC
AGCGAAAGAGCAACTACGAAACGTGGTGTGGTGGAGGTGAATTATGAAGAGGGCGCGCGATTTGAAAAGTATGTATAT
AAAAAATATATCCCGGTGTTTTATGTAGCGATAAACGAGTTTTTGATGTAAGGTATGCAGGTGTGTAAGTCTTTTGGT
TAGAAGACAAATCCAAAGTCTACTTGTGGGGATGTTCGAAGGGGAAATACTTGTATTCTATAGGTCATATCTTGTTTT
TATTGGCACAAATATAATTACATTAGCTTTTTGAGGGGGCAATAAACAGTAAACACGATGGTAATAATGGTAAAAAAA
AAAACAAGCAGTTATTTCGGATATATGTCGGCTACTCCTTGCGTCGGGCCCGAAGTCTTAGAGCCAGATATGCGAGCA
CCCGGAAGCTCACGATGAGAATGGCCAGACCATGATGAAATAACATAAGGTGGTCCCGTCGGCAAGAGACATCCACTT
AACGTATGCTTGCAATAAGTGCGAGTGAAAGGAATAGTATTCTGAGTGTCGTATTGAGTCTGAGTGAGACAGCGATAT
GATTGTTGATTAACCCTTAGCATGTCCGTGGGGTTTGAATTAACTCATAATATTAATTAGACGAAATTATTTTTAAAG
TTTTATTTTTAATAATTTGCGAGTACGCAAAGCTTCTGCATGAGCTCGGATCCAAGCTTGCATGCCTGCAGGTCGGAG
TACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTACTGTCCTCCGAGCGGAGTA
CTGTCCTCCGAGCGGAGACTCTAGCGAGCGCCGGAGTATAAATAGAGGCGCTTCGTCTACGGAGCGACAATTCAATTC
AAACAAGCAAAGTGAACACGTCGCTAAGCGAAAGCTAAGCAAATAAACAAGCGCAGCTGAACAAGCTAAACAATCTGC
AGTAAAGTGCAAGTTAAAGTGAATCAATTAAAAGTAACCAGCAACCAAGTAAATCAACTGCAACTACTGAAATCTGCC
GTACTACTTTATTGTATTCCACCAGGGCAGCCGTTCTCTGTAGGTGAA
TTGCATACGAACGTTATTCACGCTCACTTTCCTTATCATAAGACTCACAGCATAACTCAGACTCACTCTGTCGCTATA
CTAACAACTAATTGGGAATCGTACAGGCACCCCAAACTTAATTGAGTATTATAATTAATCTGCTTTAATAAAAATTTC
AAAATAAAAATTATTAAACGCTCATGCGTTTCGAAGACGTACTCGAGCCTAGGTTCGAACGTACGGACGTCCAGCCTC
ATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCATGACAGGAGGCTCGCCTCAT
GACAGGAGGCTCGCCTCTGAGATCGCTCGCGGCCTCATATTTATCTCCGCGAAGCAGATGCCTCGCTGTTAAGTTAAG
TTTGTTCGTTTCACTTGTGCAGCGATTCGCTTTCGATTCGTTTATTTGTTCGCGTCGACTTGTTCGATTTGTTAGACG
TCATTTCACGTTCAATTTCACTTAGTTAATTTTCATTGGTCGTTGGTTCATTTAGTTGACGTTGATGACTTTAGACGG
AATAACTTCGTAAATAGTCCCAATAACAGAGTACTCGCCTATGTATAAACTTACATAAATCTTTTTATTTGTTTATCC
CCAAGGCGCGTGTAAAGGGGCTTTTCACGGTGGACTGCAGATTCTTTGGTAATAATAGTACTGTAATTGGATATTTTT
ATCCGCATAGTGCTCCGGGAAAGCAGAGCGCGCAAAGCCACTACTGCCACTTTTGGAGACTGTGTACGTCGAGGGCCT
CTGCCAGTGTCGAACAGACATTCGCCTACGGCCCTCGTCTGTTCGGGCAGTCCCGCGCAGTCGCCCACAACCGCCCAC
AGCCCCGACCGAATTGATACGCCGTAGTCTCGTCTAACATGACTCTCACGTGGTATACGCCACACTTTATGGCGTGGC
TTAGCGCGCCTTGATTGCTGTCAGCGAGGTTCCAGCAGCTTGTTTTCCACTTACACAACGCCTCTCGCCCACCCTCTG
TCGCTTTCTCGTTGATGCTTTGCACCACACCACCTCCACTTAATACTTCTCCCGCGCGCTAAACTTTTCATACATATA
TTTTTTATATAGGGCCACAAAATACATCGCTATTTGCTCAAAAACTACATTCCATACGTCCACACATTCAGAAAACCA
ATCTTCTGTTTAGGTTTCAGATGAACACCCCTACAAGCTTCCCCTTTATGAACATAAGATATCCAGTATAGAACAAAA
ATAACCGTGTTTATATTAATGTAATCGAAAAACTCCCCCGTTATTTGTCATTTGTGCTACCATTATTACCATTTTTTT
TTTTGTTCGTCAATAAAGCCTATATACAGCCGATGAGGAACGCAGCCCGGGCTTCAGAATCTCGGTCTATACGCTCGT
GGGCCTTCGAGTGCTACTCTTACCGGTCTG
3 ' P
UAS sites
1873
1951
2029
2107
2185
2263
2341
2419
2497
2575
2653
2731
2809
2887
2965
3043
3121
3199
3277
AAGAAGTAATTATTGAATACAAGAAGAGAACTCTGAATAGGGAATTGGGGAATTCGTTAACAGATCTGACAAGTTTGT
ACAAAAAAGCAGGCTCCAGGAAA ATG ATC CTT CTG CTG CTG GGT GTG CTA GTG GTT CTA ATG
GCC CTA CCG CCG CCC ACC GCA GGC ACC ACC GAT TGG ATG CAG AGC TGC GGT ACA TGC
CAC TGT CAG TGG AAT TCG GGC AAG AAG AGC GCC GAC TGC AAG AAC AAG GCG CTA ACC
AAA ATT CCG CAG GAC ATG AGC AAC GAG ATG CAG GTG CTG GAC TTT GCC CAC AAT CAA
ATA CCC GAG CTG CGG CGC GAA GAG TTC CTA CTG GCC GGT CTG CCC AAT GTG CAC AAG
ATC TTT TTG CGC AAC TGC ACC ATC CAG GAG GTG CAT CGC GAG GCC TTC AAG GGT CTG
CAT ATC CTA ATC GAG CTG GAC CTG TCG GGC AAT CGG ATA CGG GAA CTG CAT CCG GGC
ACT TTC GCC GGC CTG GAG AAG CTG CGC AAC ATG ATC ATC AAC AAC AAC GAG ATC GAG
GTG CTG CCC AAC CAT CTG TTC GTC AAC CTG AGC TTC CTG TCG CGC ATC GAG TTC CGG
AAC AAT CGA TTG CGC CAG GTG CAG CTG CAC GTC TTC GCT GGC ACA ATG GCG CTG AGC
TTCTTCATTAATAACTTATGTTCTTCTCTTGAGACTTATCCCTTAACCCCTTAAGCAATTGTCTAGACTGTTCAAACA
TGTTTTTTCGTCCGAGGTCCTTT TAC TAG GAA GAC GAC GAC CCA CAC GAT CAC CAA GAT TAC
CGG GAT GGC GGC GGG TGG CGT CCG TGG TGG CTA ACC TAC GTC TCG ACG CCA TGT ACG
GTG ACA GTC ACC TTA AGC CCG TTC TTC TCG CGG CTG ACG TTC TTG TTC CGC GAT TGG
TTT TAA GGC GTC CTG TAC TCG TTG CTC TAC GTC CAC GAC CTG AAA CGG GTG TTA GTT
TAT GGG CTC GAC GCC GCG CTT CTC AAG GAT GAC CGG CCA GAC GGG TTA CAC GTG TTC
TAG AAA AAC GCG TTG ACG TGG TAG GTC CTC CAC GTA GCG CTC CGG AAG TTC CCA GAC
GTA TAG GAT TAG CTC GAC CTG GAC AGC CCG TTA GCC TAT GCC CTT GAC GTA GGC CCG
TGA AAG CGG CCG GAC CTC TTC GAC GCG TTG TAC TAG TAG TTG TTG TTG CTC TAG CTC
CAC GAC GGG TTG GTA GAC AAG CAG TTG GAC TCG AAG GAC AGC GCG TAG CTC AAG GCC
TTG TTA GCT AAC GCG GTC CAC GTC GAC GTG CAG AAG CGA CCG TGT TAC CGC GAC TCG
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N M I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P P D L L
A F P A R V P P T S P S M Q S S Q S N
I P D Q V I Y G I R S P P S L T S P V
Y T H M T P H G I Y G T K T M T A P H
N G F M T L Q H P K S R N L A L I A T
T N S S R Q H Q H H H Q L Q Q Q Q Q H
H H H H Q Q Q Q Q Q Q Q Q Q Q H P L A
T T S P F L P A P V V Y S P A T G V E
P V Y D N L G L R T T A G G N S T L N
L T K I A G S Q G G A G Q Q Y S M R D
R P L P A T P S L T S V S S A T N A S
K I Y E P I H E L I Q Q Q Q Q L Q Q Q
Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G
H A A Y L S P G S G A A V S P S H A S
S S G D S P K A A K I P P R P P P K P
K K K M S V T T T R S G Q G S T S Q L
F D D E G E D G T E V D P A F L Y K V
V V P R A R D P P V A T M V S K G E E
L F T G V V P I L V E L D G D V N G H
K F S V S G E G E G D A T Y G K L T L
K F I C T T G K L P V P W P T L V T T
L T Y G V Q C F S R Y P D H M K Q H D
F F K S A M P E G Y V Q E R T I F F K
D D G N Y K T R A E V K F E G D T L V
N R I E L K G I D F K E D G N I L G H
K L E Y N Y N S H N V Y I M A D K Q K
N G I K V N F K I R H N I E D G S V Q
L A D H Y Q Q N T P I G D G P V L L P
D N H Y L S T Q S A L S K D P N E K R
D H M V L L E F V T A A G I T L G M D
E L Y K •
EcoR1
P o l y l i n k e r
HpaI BglII attB1
K5signal sequence
KEK5
Start of Good Sequence
N FLANK
LRRs
BglII
originally C
oligo238
3355
3433
3495
3552
3609
3666
3723
3780
3837
3894
3951
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
GCC ATT TCG CTG GAA CAG AAC CGC CTC TCA CAT CTG CAC AAG GAG ACA TTC AAG GAT
CTG CAG AAG CTG ATG CAT CTA TCG CTG CAG GGT AAC GCA TGG AAC TGC AGC TGC GAG
CTG CAG GAC TTT CGC GAC TTT GCG ATC AGC AAA CGG CTC TAC ACA CCG CCC ACC GAT
TGC CAG GAG CCG CCA CAG CTG CGC GGC AAG CTG TGG AGC GAG GTG CCA TCG GAG AAC
TTC GCC TGC CGG CCG CGC ATT TTG GGT TCC GTG CGC TCC TTC ATC GAG GCC AAT CAC
GAC AAT ATC TCG CTA CCC TGC CGC ATT GTC GGC AGT CCG CGT CCC AAT GTC ACC TGG
GTG TAC AAC AAG CGG CCA TTG CAG CAG TAC GAC CCG CGT GTG CGT GTC CTC ACC TCC
GTG GAA CAG ATG CCG GAG CAG CCC TCC CAG GTG CTC ACC TCG GAG CTG CGC ATC GTG
GGC GTA CGG GCC TCC GAC AAG GGT GCC TAC ACC TGT GTG GCG GAT AAC CGG GGC GGA
CGG GCG GAG GCC GAG TTC CAG CTG CTC GTG AGC GGT GAC TAT GCC GGC GCG GTA TCC
GCC TCC GAT GGC ATG GGC ATG GGC GCC ATT GGG GCA CCA ACC ATT GAT CCG CAA ACG
AAC ATG TTT CTC ATC ATC TGT CTA ATC ATT ACG ACG CTG CTG CTC CTG CTG CTC GTG
CGG TAA AGC GAC CTT GTC TTG GCG GAG AGT GTA GAC GTG TTC CTC TGT AAG TTC CTA
GAC GTC TTC GAC TAC GTA GAT AGC GAC GTC CCA TTG CGT ACC TTG ACG TCG ACG CTC
GAC GTC CTG AAA GCG CTG AAA CGC TAG TCG TTT GCC GAG ATG TGT GGC GGG TGG CTA
ACG GTC CTC GGC GGT GTC GAC GCG CCG TTC GAC ACC TCG CTC CAC GGT AGC CTC TTG
AAG CGG ACG GCC GGC GCG TAA AAC CCA AGG CAC GCG AGG AAG TAG CTC CGG TTA GTG
CTG TTA TAG AGC GAT GGG ACG GCG TAA CAG CCG TCA GGC GCA GGG TTA CAG TGG ACC
CAC ATG TTG TTC GCC GGT AAC GTC GTC ATG CTG GGC GCA CAC GCA CAG GAG TGG AGG
CAC CTT GTC TAC GGC CTC GTC GGG AGG GTC CAC GAG TGG AGC CTC GAC GCG TAG CAC
CCG CAT GCC CGG AGG CTG TTC CCA CGG ATG TGG ACA CAC CGC CTA TTG GCC CCG CCT
GCC CGC CTC CGG CTC AAG GTC GAC GAG CAC TCG CCA CTG ATA CGG CCG CGC CAT AGG
CGG AGG CTA CCG TAC CCG TAC CCG CGG TAA CCC CGT GGT TGG TAA CTA GGC GTT TGC
TTG TAC AAA GAG TAG TAG ACA GAT TAG TAA TGC TGC GAC GAC GAG GAC GAC GAG CAC
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N M I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P P D L L
A F P A R V P P T S P S M Q S S Q S N
I P D Q V I Y G I R S P P S L T S P V
Y T H M T P H G I Y G T K T M T A P H
N G F M T L Q H P K S R N L A L I A T
T N S S R Q H Q H H H Q L Q Q Q Q Q H
H H H H Q Q Q Q Q Q Q Q Q Q Q H P L A
T T S P F L P A P V V Y S P A T G V E
P V Y D N L G L R T T A G G N S T L N
L T K I A G S Q G G A G Q Q Y S M R D
R P L P A T P S L T S V S S A T N A S
K I Y E P I H E L I Q Q Q Q Q L Q Q Q
Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G
H A A Y L S P G S G A A V S P S H A S
S S G D S P K A A K I P P R P P P K P
K K K M S V T T T R S G Q G S T S Q L
F D D E G E D G T E V D P A F L Y K V
V V P R A R D P P V A T M V S K G E E
L F T G V V P I L V E L D G D V N G H
K F S V S G E G E G D A T Y G K L T L
K F I C T T G K L P V P W P T L V T T
L T Y G V Q C F S R Y P D H M K Q H D
F F K S A M P E G Y V Q E R T I F F K
D D G N Y K T R A E V K F E G D T L V
N R I E L K G I D F K E D G N I L G H
K L E Y N Y N S H N V Y I M A D K Q K
N G I K V N F K I R H N I E D G S V Q
L A D H Y Q Q N T P I G D G P V L L P
D N H Y L S T Q S A L S K D P N E K R
D H M V L L E F V T A A G I T L G M D
E L Y K •
oligo594
C FLANK
Ig
end Ig?
Tm
4008
4065
4122
4179
4236
4293
4350
4407
4464
4521
4578
4635
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
GCG GTG CTG ACG CTC TTC TGG TAC TGC CGT CGC ATC AAG ACC TAT CAA AAG GAC ACC
ACC ATG ATG AGC GGC GAC GGG CTG ATC TCT TCC AAG ATG GAC AAG ACG CAC AAC GGC
TCC ATG CTC GAG GGT TCC GTC ATC ATG GAG ATG CAG AAG AGC CTG CTC AAC GAG GTC
AAT CCA GTC GAG AAG CCG CCA CGG CGC ACG GAC ATC GAG AGC GTG GAT GGT GGC GAT
GAC GTG CTC GAG ATC AAG AAG ACG CTG CTC GAC GAC ACC GTC TAT GTG GCC AAT CAC
TCG CGC GAC GAA GAA GCC GTC TCA GTG GCC ATG TCG GAT ACG ACG ACC ACG CCC CGA
TCT CGA CAC ACC TAC GTG GAT GAT GCG TAT GCC AAT AGC TTG CCA CCG GAT CTG CTG
GCC TTT CCC GCT CGC GTG CCG CCC ACC TCG CCC TCG ATG CAA TCG TCG CAG TCG AAC
ATA CCC GAC CAG GTG ATC TAC GGC ATC CGT TCG CCA CCG TCG CTA ACC AGT CCG GTC
TAC ACG CAT ATG ACG CCG CAC GGC ATC TAC GGC ACC AAG ACG ATG ACG GCT CCG CAT
AAC GGC TTT ATG ACG CTG CAG CAT CCC AAG TCG CGC AAC CTG GCG CTC ATT GCC ACC
CGC CAC GAC TGC GAG AAG ACC ATG ACG GCA GCG TAG TTC TGG ATA GTT TTC CTG TGG
TGG TAC TAC TCG CCG CTG CCC GAC TAG AGA AGG TTC TAC CTG TTC TGC GTG TTG CCG
AGG TAC GAG CTC CCA AGG CAG TAG TAC CTC TAC GTC TTC TCG GAC GAG TTG CTC CAG
TTA GGT CAG CTC TTC GGC GGT GCC GCG TGC CTG TAG CTC TCG CAC CTA CCA CCG CTA
CTG CAC GAG CTC TAG TTC TTC TGC GAC GAG CTG CTG TGG CAG ATA CAC CGG TTA GTG
AGC GCG CTG CTT CTT CGG CAG AGT CAC CGG TAC AGC CTA TGC TGC TGG TGC GGG GCT
AGA GCT GTG TGG ATG CAC CTA CTA CGC ATA CGG TTA TCG AAC GGT GGC CTA GAC GAC
CGG AAA GGG CGA GCG CAC GGC GGG TGG AGC GGG AGC TAC GTT AGC AGC GTC AGC TTG
TAT GGG CTG GTC CAC TAG ATG CCG TAG GCA AGC GGT GGC AGC GAT TGG TCA GGC CAG
ATG TGC GTA TAC TGC GGC GTG CCG TAG ATG CCG TGG TTC TGC TAC TGC CGA GGC GTA
TTG CCG AAA TAC TGC GAC GTC GTA GGG TTC AGC GCG TTG GAC CGC GAG TAA CGG TGG
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N M I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P P D L L
A F P A R V P P T S P S M Q S S Q S N
I P D Q V I Y G I R S P P S L T S P V
Y T H M T P H G I Y G T K T M T A P H
N G F M T L Q H P K S R N L A L I A T
T N S S R Q H Q H H H Q L Q Q Q Q Q H
H H H H Q Q Q Q Q Q Q Q Q Q Q H P L A
T T S P F L P A P V V Y S P A T G V E
P V Y D N L G L R T T A G G N S T L N
L T K I A G S Q G G A G Q Q Y S M R D
R P L P A T P S L T S V S S A T N A S
K I Y E P I H E L I Q Q Q Q Q L Q Q Q
Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G
H A A Y L S P G S G A A V S P S H A S
S S G D S P K A A K I P P R P P P K P
K K K M S V T T T R S G Q G S T S Q L
F D D E G E D G T E V D P A F L Y K V
V V P R A R D P P V A T M V S K G E E
L F T G V V P I L V E L D G D V N G H
K F S V S G E G E G D A T Y G K L T L
K F I C T T G K L P V P W P T L V T T
L T Y G V Q C F S R Y P D H M K Q H D
F F K S A M P E G Y V Q E R T I F F K
D D G N Y K T R A E V K F E G D T L V
N R I E L K G I D F K E D G N I L G H
K L E Y N Y N S H N V Y I M A D K Q K
N G I K V N F K I R H N I E D G S V Q
L A D H Y Q Q N T P I G D G P V L L P
D N H Y L S T Q S A L S K D P N E K R
D H M V L L E F V T A A G I T L G M D
E L Y K •
IC oligo745 apK5_seq_int_5'
IC1 modified oligo746 apK5_seq_int_3'
∆1 ConfirmedIC1
oligo240
∆234 ConfirmedIC2 modified
IC2
4692
4749
4806
4863
4920
4977
5034
5091
5148
5205
5262
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
ACC AAC AGC AGT CGC CAG CAC CAG CAC CAC CAT CAG CTG CAG CAG CAG CAG CAG CAC
CAC CAC CAC CAC CAG CAG CAA CAA CAA CAG CAG CAG CAG CAG CAA CAT CCG CTG GCC
ACC ACA TCG CCC TTC CTG CCC GCA CCC GTC GTC TAT TCG CCG GCC ACG GGT GTG GAG
CCA GTG TAC GAC AAC TTG GGA TTG CGA ACC ACT GCC GGC GGC AAC TCC ACC CTC AAT
CTG ACC AAG ATC GCC GGC TCA CAG GGG GGC GCT GGT CAG CAG TAC TCG ATG CGG GAC
CGA CCA CTT CCG GCC ACG CCC AGC CTG ACA TCG GTG TCC TCG GCG ACC AAT GCC AGT
AAG ATT TAC GAG CCC ATA CAC GAG CTG ATT CAG CAG CAA CAG CAG TTG CAA CAA CAA
CAA CAG CAG CAG CAG CAG CGA CTG GGC TCC ATG GAC ACG GAA CCC CTG TAC GGA GTT
CGG CAA CAG GGG ATC ACG ATA CTG CCC GGC TCG AGC ATT AGC GGT GCC GGA CTG GGC
CAC GCC GCC TAC CTT TCA CCC GGC TCG GGT GCC GCC GTA TCG CCA AGC CAC GCC AGC
AGC AGC GGT GAC TCT CCG AAG GCC GCC AAG ATC CCA CCA CGC CCA CCA CCG AAG CCC
AAG AAG AAG ATG TCC GTG ACG ACG ACG CGC AGC GGC CAG GGC AGC ACC AGC CAG CTC
TGG TTG TCG TCA GCG GTC GTG GTC GTG GTG GTA GTC GAC GTC GTC GTC GTC GTC GTG
GTG GTG GTG GTG GTC GTC GTT GTT GTT GTC GTC GTC GTC GTC GTT GTA GGC GAC CGG
TGG TGT AGC GGG AAG GAC GGG CGT GGG CAG CAG ATA AGC GGC CGG TGC CCA CAC CTC
GGT CAC ATG CTG TTG AAC CCT AAC GCT TGG TGA CGG CCG CCG TTG AGG TGG GAG TTA
GAC TGG TTC TAG CGG CCG AGT GTC CCC CCG CGA CCA GTC GTC ATG AGC TAC GCC CTG
GCT GGT GAA GGC CGG TGC GGG TCG GAC TGT AGC CAC AGG AGC CGC TGG TTA CGG TCA
TTC TAA ATG CTC GGG TAT GTG CTC GAC TAA GTC GTC GTT GTC GTC AAC GTT GTT GTT
GTT GTC GTC GTC GTC GTC GCT GAC CCG AGG TAC CTG TGC CTT GGG GAC ATG CCT CAA
GCC GTT GTC CCC TAG TGC TAT GAC GGG CCG AGC TCG TAA TCG CCA CGG CCT GAC CCG
GTG CGG CGG ATG GAA AGT GGG CCG AGC CCA CGG CGG CAT AGC GGT TCG GTG CGG TCG
TCG TCG CCA CTG AGA GGC TTC CGG CGG TTC TAG GGT GGT GCG GGT GGT GGC TTC GGG
TTC TTC TTC TAC AGG CAC TGC TGC TGC GCG TCG CCG GTC CCG TCG TGG TCG GTC GAG
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N M I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P P D L L
A F P A R V P P T S P S M Q S S Q S N
I P D Q V I Y G I R S P P S L T S P V
Y T H M T P H G I Y G T K T M T A P H
N G F M T L Q H P K S R N L A L I A T
T N S S R Q H Q H H H Q L Q Q Q Q Q H
H H H H Q Q Q Q Q Q Q Q Q Q Q H P L A
T T S P F L P A P V V Y S P A T G V E
P V Y D N L G L R T T A G G N S T L N
L T K I A G S Q G G A G Q Q Y S M R D
R P L P A T P S L T S V S S A T N A S
K I Y E P I H E L I Q Q Q Q Q L Q Q Q
Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G
H A A Y L S P G S G A A V S P S H A S
S S G D S P K A A K I P P R P P P K P
K K K M S V T T T R S G Q G S T S Q L
F D D E G E D G T E V D P A F L Y K V
V V P R A R D P P V A T M V S K G E E
L F T G V V P I L V E L D G D V N G H
K F S V S G E G E G D A T Y G K L T L
K F I C T T G K L P V P W P T L V T T
L T Y G V Q C F S R Y P D H M K Q H D
F F K S A M P E G Y V Q E R T I F F K
D D G N Y K T R A E V K F E G D T L V
N R I E L K G I D F K E D G N I L G H
K L E Y N Y N S H N V Y I M A D K Q K
N G I K V N F K I R H N I E D G S V Q
L A D H Y Q Q N T P I G D G P V L L P
D N H Y L S T Q S A L S K D P N E K R
D H M V L L E F V T A A G I T L G M D
E L Y K •
∆ IC3CLIC4
IC5
5319
5376
5433
5490
5547
5604
5661
5718
5775
5832
5889
5946
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
TTC GAC GAC GAG GGC GAG GAT GGC ACC GAG GTC GAC CCA GCT TTC TTG TAC AAA GTG
GTG GTA CCG CGG GCC CGG GAT CCA CCG GTC GCC ACC ATG GTG AGC AAG GGC GAG GAG
CTG TTC ACC GGG GTG GTG CCC ATC CTG GTC GAG CTG GAC GGC GAC GTA AAC GGC CAC
AAG TTC AGC GTG TCC GGC GAG GGC GAG GGC GAT GCC ACC TAC GGC AAG CTG ACC CTG
AAG TTC ATC TGC ACC ACC GGC AAG CTG CCC GTG CCC TGG CCC ACC CTC GTG ACC ACC
CTG ACC TAC GGC GTG CAG TGC TTC AGC CGC TAC CCC GAC CAC ATG AAG CAG CAC GAC
TTC TTC AAG TCC GCC ATG CCC GAA GGC TAC GTC CAG GAG CGC ACC ATC TTC TTC AAG
GAC GAC GGC AAC TAC AAG ACC CGC GCC GAG GTG AAG TTC GAG GGC GAC ACC CTG GTG
AAC CGC ATC GAG CTG AAG GGC ATC GAC TTC AAG GAG GAC GGC AAC ATC CTG GGG CAC
AAG CTG GAG TAC AAC TAC AAC AGC CAC AAC GTC TAT ATC ATG GCC GAC AAG CAG AAG
AAC GGC ATC AAG GTG AAC TTC AAG ATC CGC CAC AAC ATC GAG GAC GGC AGC GTG CAG
CTC GCC GAC CAC TAC CAG CAG AAC ACC CCC ATC GGC GAC GGC CCC GTG CTG CTG CCC
AAG CTG CTG CTC CCG CTC CTA CCG TGG CTC CAG CTG GGT CGA AAG AAC ATG TTT CAC
CAC CAT GGC GCC CGG GCC CTA GGT GGC CAG CGG TGG TAC CAC TCG TTC CCG CTC CTC
GAC AAG TGG CCC CAC CAC GGG TAG GAC CAG CTC GAC CTG CCG CTG CAT TTG CCG GTG
TTC AAG TCG CAC AGG CCG CTC CCG CTC CCG CTA CGG TGG ATG CCG TTC GAC TGG GAC
TTC AAG TAG ACG TGG TGG CCG TTC GAC GGG CAC GGG ACC GGG TGG GAG CAC TGG TGG
GAC TGG ATG CCG CAC GTC ACG AAG TCG GCG ATG GGG CTG GTG TAC TTC GTC GTG CTG
AAG AAG TTC AGG CGG TAC GGG CTT CCG ATG CAG GTC CTC GCG TGG TAG AAG AAG TTC
CTG CTG CCG TTG ATG TTC TGG GCG CGG CTC CAC TTC AAG CTC CCG CTG TGG GAC CAC
TTG GCG TAG CTC GAC TTC CCG TAG CTG AAG TTC CTC CTG CCG TTG TAG GAC CCC GTG
TTC GAC CTC ATG TTG ATG TTG TCG GTG TTG CAG ATA TAG TAC CGG CTG TTC GTC TTC
TTG CCG TAG TTC CAC TTG AAG TTC TAG GCG GTG TTG TAG CTC CTG CCG TCG CAC GTC
GAG CGG CTG GTG ATG GTC GTC TTG TGG GGG TAG CCG CTG CCG GGG CAC GAC GAC GGG
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N M I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P P D L L
A F P A R V P P T S P S M Q S S Q S N
I P D Q V I Y G I R S P P S L T S P V
Y T H M T P H G I Y G T K T M T A P H
N G F M T L Q H P K S R N L A L I A T
T N S S R Q H Q H H H Q L Q Q Q Q Q H
H H H H Q Q Q Q Q Q Q Q Q Q Q H P L A
T T S P F L P A P V V Y S P A T G V E
P V Y D N L G L R T T A G G N S T L N
L T K I A G S Q G G A G Q Q Y S M R D
R P L P A T P S L T S V S S A T N A S
K I Y E P I H E L I Q Q Q Q Q L Q Q Q
Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G
H A A Y L S P G S G A A V S P S H A S
S S G D S P K A A K I P P R P P P K P
K K K M S V T T T R S G Q G S T S Q L
F D D E G E D G T E V D P A F L Y K V
V V P R A R D P P V A T M V S K G E E
L F T G V V P I L V E L D G D V N G H
K F S V S G E G E G D A T Y G K L T L
K F I C T T G K L P V P W P T L V T T
L T Y G V Q C F S R Y P D H M K Q H D
F F K S A M P E G Y V Q E R T I F F K
D D G N Y K T R A E V K F E G D T L V
N R I E L K G I D F K E D G N I L G H
K L E Y N Y N S H N V Y I M A D K Q K
N G I K V N F K I R H N I E D G S V Q
L A D H Y Q Q N T P I G D G P V L L P
D N H Y L S T Q S A L S K D P N E K R
D H M V L L E F V T A A G I T L G M D
E L Y K •
End of Good SequenceIC6 (PDZ)
attB2
KpnI oligo 445 T7
EGFPN1 polyl inker
XmaI
EGFPN1
#54
6003
6060
6117
6174
6231
6288
6345
6402
6459
6516
6573
6630
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
GAC AAC CAC TAC CTG AGC ACC CAG TCC GCC CTG AGC AAA GAC CCC AAC GAG AAG CGC
GAT CAC ATG GTC CTG CTG GAG TTC GTG ACC GCC GCC GGG ATC ACT CTC GGC ATG GAC
GAG CTG TAC AAG TAA AGCGGCCGCGACTCTAGAGGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATA
ATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACT
GATTCTAATTGTTTGTGTATTTTAGATTCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTTTAATGA
GGAAAACCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTCC
AAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGCTAAGTTTTTTGAGTCATGCTGTGTTTAG
TAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCACTGCTATACAAGAAAATTATGGAAAA
ATATTCTGTAACCTTTATAAGTAGGCATAACAGTTATAATCATAACATACTGTTTTTTCTTACTCCACACAGGCATAG
AGTGTCTGCTATTAATAACTATGCTCAAAAATTGTGTACCTTTAGCTTTTTAATTTGTAAAGGGGTTAATAAGGAATA
TTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACC
TCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATG
GTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCA
AACTCATCAATGTATCTTATCATGTCTGGATCGGATCCACTAGAAGGCCTTAGTATGTATGTAAGTTAATAAAACCCT
TTTTTGGAGAATGTAGATTTAAAAAAACATATTTTTTTTTTATTTTTTACTGCACTGGACATCATTGAACTTATCTGA
CTG TTG GTG ATG GAC TCG TGG GTC AGG CGG GAC TCG TTT CTG GGG TTG CTC TTC GCG
CTA GTG TAC CAG GAC GAC CTC AAG CAC TGG CGG CGG CCC TAG TGA GAG CCG TAC CTG
CTC GAC ATG TTC ATT TCGCCGGCGCTGAGATCTCCTAGAAACACTTCCTTGGAATGAAGACACCACACTGTAT
TAACCTGTTTGATGGATGTCTCTAAATTTCGAGATTCCATTTATATTTTAAAAATTCACATATTACACAATTTGATGA
CTAAGATTAACAAACACATAAAATCTAAGGTTGGATACCTTGACTACTTACCCTCGTCACCACCTTACGGAAATTACT
CCTTTTGGACAAAACGAGTCTTCTTTACGGTAGATCACTACTACTCCGATGACGACTGAGAGTTGTAAGATGAGGAGG
TTTTTTCTTCTCTTTCCATCTTCTGGGGTTCCTGAAAGGAAGTCTTAACGATTCAAAAAACTCAGTACGACACAAATC
ATTATCTTGAGAACGAACGAAACGATAAATGTGGTGTTTCCTTTTTCGACGTGACGATATGTTCTTTTAATACCTTTT
TATAAGACATTGGAAATATTCATCCGTATTGTCAATATTAGTATTGTATGACAAAAAAGAATGAGGTGTGTCCGTATC
TCACAGACGATAATTATTGATACGAGTTTTTAACACATGGAAATCGAAAAATTAAACATTTCCCCAATTATTCCTTAT
AAACTACATATCACGGAACTGATCTCTAGTATTAGTCGGTATGGTGTAAACATCTCCAAAATGAACGAAATTTTTTGG
AGGGTGTGGAGGGGGACTTGGACTTTGTATTTTACTTACGTTAACAACAACAATTGAACAAATAACGTCGAATATTAC
CAATGTTTATTTCGTTATCGTAGTGTTTAAAGTGTTTATTTCGTAAAAAAAGTGACGTAAGATCAACACCAAACAGGT
TTGAGTAGTTACATAGAATAGTACAGACCTAGCCTAGGTGATCTTCCGGAATCATACATACATTCAATTATTTTGGGA
AAAAACCTCTTACATCTAAATTTTTTTGTATAAAAAAAAAATAAAAAATGACGTGACCTGTAGTAACTTGAATAGACT
M I L L L L G V L V V L M
A L P P P T A G T T D W M Q S C G T C
H C Q W N S G K K S A D C K N K A L T
K I P Q D M S N E M Q V L D F A H N Q
I P E L R R E E F L L A G L P N V H K
I F L R N C T I Q E V H R E A F K G L
H I L I E L D L S G N R I R E L H P G
T F A G L E K L R N M I I N N N E I E
V L P N H L F V N L S F L S R I E F R
N N R L R Q V Q L H V F A G T M A L S
A I S L E Q N R L S H L H K E T F K D
L Q K L M H L S L Q G N A W N C S C E
L Q D F R D F A I S K R L Y T P P T D
C Q E P P Q L R G K L W S E V P S E N
F A C R P R I L G S V R S F I E A N H
D N I S L P C R I V G S P R P N V T W
V Y N K R P L Q Q Y D P R V R V L T S
V E Q M P E Q P S Q V L T S E L R I V
G V R A S D K G A Y T C V A D N R G G
R A E A E F Q L L V S G D Y A G A V S
A S D G M G M G A I G A P T I D P Q T
N M F L I I C L I I T T L L L L L L V
A V L T L F W Y C R R I K T Y Q K D T
T M M S G D G L I S S K M D K T H N G
S M L E G S V I M E M Q K S L L N E V
N P V E K P P R R T D I E S V D G G D
D V L E I K K T L L D D T V Y V A N H
S R D E E A V S V A M S D T T T T P R
S R H T Y V D D A Y A N S L P P D L L
A F P A R V P P T S P S M Q S S Q S N
I P D Q V I Y G I R S P P S L T S P V
Y T H M T P H G I Y G T K T M T A P H
N G F M T L Q H P K S R N L A L I A T
T N S S R Q H Q H H H Q L Q Q Q Q Q H
H H H H Q Q Q Q Q Q Q Q Q Q Q H P L A
T T S P F L P A P V V Y S P A T G V E
P V Y D N L G L R T T A G G N S T L N
L T K I A G S Q G G A G Q Q Y S M R D
R P L P A T P S L T S V S S A T N A S
K I Y E P I H E L I Q Q Q Q Q L Q Q Q
Q Q Q Q Q Q R L G S M D T E P L Y G V
R Q Q G I T I L P G S S I S G A G L G
H A A Y L S P G S G A A V S P S H A S
S S G D S P K A A K I P P R P P P K P
K K K M S V T T T R S G Q G S T S Q L
F D D E G E D G T E V D P A F L Y K V
V V P R A R D P P V A T M V S K G E E
L F T G V V P I L V E L D G D V N G H
K F S V S G E G E G D A T Y G K L T L
K F I C T T G K L P V P W P T L V T T
L T Y G V Q C F S R Y P D H M K Q H D
F F K S A M P E G Y V Q E R T I F F K
D D G N Y K T R A E V K F E G D T L V
N R I E L K G I D F K E D G N I L G H
K L E Y N Y N S H N V Y I M A D K Q K
N G I K V N F K I R H N I E D G S V Q
L A D H Y Q Q N T P I G D G P V L L P
D N H Y L S T Q S A L S K D P N E K R
D H M V L L E F V T A A G I T L G M D
E L Y K •
Xba I SV40 Poly A
HpaI
B a m H I
white gene
6687
6744
6801
6874
6952
7030
7108
7186
7264
7342
7420
7498
7576
7654
7732
1
14
33
52
71
90
109
128
147
166
185
204
223
242
261
280
299
318
337
356
375
394
413
432
451
470
489
508
527
546
565
584
603
622
641
660
679
698
717
736
755
774
793
812
831
850
869
888
907
926
945
964
983
1002
1021
1040
1059
1078
1097
1116
TCAGTTTTAAATTTACTTCGATCCAAGGGTATTTGAAGTACCAGGTTCTTTCGATTACCTCTCACTCAAAATGACATT
CCACTCAAAGTCAGCGCTGTTTGCCTCCTTCTCTGTCCACAGAAATATCGCCGTCTCTTTCGCCGCTGCGTCCGCTAT
CTCTTTCGCCACCGTTTGTAGCGTTACCTAGCGTCAATGTCCGCCTTCAGTTGCACTTTGTCAGCGGTTTCGTGACGA
AGCTCCAAGCGGTTTACGCCATCAATTAAACACAAAGTGCTGTGCCAAAACTCCTCTCGCTTCTTATTTTTGTTTGTT
TTTTGAGTGATTGGGGTGGTGATTGGTTTTGGGTGGGTAAGCAGGGGAAAGTGTGAAAAATCCCGGCAATGGGCCAAG
AGGATCAGGAGCTATTAATTCGCGGAGGCAGCAAACACCCATCTGCCGAGCATCTGAACAATGTGAGTAGTACATGTG
CATACATCTTAAGTTCACTTGATCTATAGGAACTGCGATTGCAACATCAAATTGTCTGCGGCGTGAGAACTGCGACCC
ACAAAAATCCCAAACCGCAATCGCACAAACAAATAGTGACACGAAACAGATTATTCTGGTAGCTGTGCTCGCTATATA
AGACAATTTTTAAGATCATATCATGATCAAGACATCTAAAGGCATTCATTTTCGACTACATTCTTTTTTACAAAAAAT
ATAACAACCAGATATTTTAAGCTGATCCTAGATGCACAAAAAATAAATAAAAGTATAAACCTACTTCGTAGGATACTT
CGTTTTGTTCGGGGTTAGATGAGCATAACGCTTGTAGTTGATATTTGAGATCCCCTATCATTGCAGGGTGACAGCGGA
GCGGCTTCGCAGAGCTGCATTAACCAGGGCTTCGGGCAGGCCAAAAACTACGGCACGCTCCTGCCACCCAGTCCGCCG
GAGGACTCCGGTTCAGGGAGCGGCCAACTAGCCGAGAACCTCACCTATGCCTGGCACAATATGGACATCTTTGGGGCG
GTCAATCAGCCGGGCTCCGGATGGCGGCAGCTGGTCAACCGGACACGCGGACTATTCTGCAACGAGCGACACATACCG
GCGCCCAGGAAACATTTGCTCAAGAACGGTGAGTTTCTATTCGCAGTCGGCTGATCTGTGTGAAATCTTAATAAAGGG
TCCAATTACCAATTTGAAACTCAGTTTGCGGCGTGGCCTATCCGGGCGAACTTTTGGCCGTGATGGGCAGTTCCGGTG
CCGGAAAGACGACCCTGCTGAATGCCCTTGCCTTTCGATCGCCGCAGGGCATCCAAGTATCGCCATCCGGGATGCGAC
AGTCAAAATTTAAATGAAGCTAGGTTCCCATAAACTTCATGGTCCAAGAAAGCTAATGGAGAGTGAGTTTTACTGTAA
GGTGAGTTTCAGTCGCGACAAACGGAGGAAGAGACAGGTGTCTTTATAGCGGCAGAGAAAGCGGCGACGCAGGCGATA
GAGAAAGCGGTGGCAAACATCGCAATGGATCGCAGTTACAGGCGGAAGTCAACGTGAAACAGTCGCCAAAGCACTGCT
TCGAGGTTCGCCAAATGCGGTAGTTAATTTGTGTTTCACGACACGGTTTTGAGGAGAGCGAAGAATAAAAACAAACAA
AAAACTCACTAACCCCACCACTAACCAAAACCCACCCATTCGTCCCCTTTCACACTTTTTAGGGCCGTTACCCGGTTC
TCCTAGTCCTCGATAATTAAGCGCCTCCGTCGTTTGTGGGTAGACGGCTCGTAGACTTGTTACACTCATCATGTACAC
GTATGTAGAATTCAAGTGAACTAGATATCCTTGACGCTAACGTTGTAGTTTAACAGACGCCGCACTCTTGACGCTGGG
TGTTTTTAGGGTTTGGCGTTAGCGTGTTTGTTTATCACTGTGCTTTGTCTAATAAGACCATCGACACGAGCGATATAT
TCTGTTAAAAATTCTAGTATAGTACTAGTTCTGTAGATTTCCGTAAGTAAAAGCTGATGTAAGAAAAAATGTTTTTTA
TATTGTTGGTCTATAAAATTCGACTAGGATCTACGTGTTTTTTATTTATTTTCATATTTGGATGAAGCATCCTATGAA
GCAAAACAAGCCCCAATCTACTCGTATTGCGAACATCAACTATAAACTCTAGGGGATAGTAACGTCCCACTGTCGCCT
CGCCGAAGCGTCTCGACGTAATTGGTCCCGAAGCCCGTCCGGTTTTTGATGCCGTGCGAGGACGGTGGGTCAGGCGGC
CTCCTGAGGCCAAGTCCCTCGCCGGTTGATCGGCTCTTGGAGTGGATACGGACCGTGTTATACCTGTAGAAACCCCGC
CAGTTAGTCGGCCCGAGGCCTACCGCCGTCGACCAGTTGGCCTGTGCGCCTGATAAGACGTTGCTCGCTGTGTATGGC
CGCGGGTCCTTTGTAAACGAGTTCTTGCCACTCAAAGATAAGCGTCAGCCGACTAGACACACTTTAGAATTATTTCCC
AGGTTAATGGTTAAACTTTGAGTCAAACGCCGCACCGGATAGGCCCGCTTGAAAACCGGCACTACCCGTCAAGGCCAC
GGCCTTTCTGCTGGGACGACTTACGGGAACGGAAAGCTAGCGGCGTCCCGTAGGTTCATAGCGGTAGGCCCTACGCTG
7810
7888
7966
8044
8122
8200
8278
8356
8434
8512
8590
8668
8746
8824
8902
8980
9058
TGCTCAATGGCCAACCTGTGGACGCCAAGGAGATGCAGGCCAGGTGCGCCTATGTCCAGCAGGATGACCTCTTTATCG
GCTCCCTAACGGCCAGGGAACACCTGATTTTCCAGGCCATGGTGCGGATGCCACGACATCTGACCTATCGGCAGCGAG
TGGCCCGCGTGGATCAGGTGATCCAGGAGCTTTCGCTCAGCAAATGTCAGCACACGATCATCGGTGTGCCCGGCAGGG
TGAAAGGTCTGTCCGGCGGAGAAAGGAAGCGTCTGGCATTCGCCTCCGAGGCACTAACCGATCCGCCGCTTCTGATCT
GCGATGAGCCCACCTCCGGACTGGACTCATTTACCGCCCACAGCGTCGTCCAGGTGCTGAAGAAGCTGTCGCAGAAGG
GCAAGACCGTCATCCTGACCATTCATCAGCCGTCTTCCGAGCTGTTTGAGCTCTTTGACAAGATCCTTCTGATGGCCG
AGGGCAGGGTAGCTTTCTTGGGCACTCCCAGCGAAGCCGTCGACTTCTTTTCCTAGTGAGTTCGATGTGTTTATTAAG
GGTATCTAGCATTACATTACATCTCAACTCCTATCCAGCGTGGGTGCCCAGTGTCCTACCAACTACAATCCGGCGGAC
TTTTACGTACAGGTGTTGGCCGTTGTGCCCGGACGGGAGATCGAGTCCCGTGATCGGATCGCCAAGATATGCGACAAT
TTTGCTATTAGCAAAGTAGCCCGGGATATGGAGCAGTTGTTGGCCACCAAAAATTTGGAGAAGCCACTGGAGCAGCCG
GAGAATGGGTACACCTACAAGGCCACCTGGTTCATGCAGTTCCGGGCGGTCCTGTGGCGATCCTGGCTGTCGGTGCTC
AAGGAACCACTCCTCGTAAAAGTGCGACTTATTCAGACAACGGTGAGTGGTTCCAGTGGAAACAAATGATATAACGCT
TACAATTCTTGGAAACAAATTCGCTAGATTTTAGTTAGAATTGCCTGATTCCACACCCTTCTTAGTTTTTTTCAATGA
GATGTATAGTTTATAGTTTTGCAGAAAATAAATAAATTTCATTTAACTCGCGAACATGTTGAAGATATGAATATTAAT
GAGATGCGAGTAACATTTTAATTTGCAGATGGTTGCCATCTTGATTGGCCTCATCTTTTTGGGCCAACAACTCACGCA
AGTGGGCGTGATGAATATCAACGGAGCCATCTTCCTCTTCCTGACCAACATGACCTTTCAAAACGTCTTTGCCACGAT
AAATGTAAGTCTTGTTTAGAATACATTTGCATATTAATAATTTACTAACTTTCTAATGAATCGATTCGATTTAGGTGT
ACGAGTTACCGGTTGGACACCTGCGGTTCCTCTACGTCCGGTCCACGCGGATACAGGTCGTCCTACTGGAGAAATAGC
CGAGGGATTGCCGGTCCCTTGTGGACTAAAAGGTCCGGTACCACGCCTACGGTGCTGTAGACTGGATAGCCGTCGCTC
ACCGGGCGCACCTAGTCCACTAGGTCCTCGAAAGCGAGTCGTTTACAGTCGTGTGCTAGTAGCCACACGGGCCGTCCC
ACTTTCCAGACAGGCCGCCTCTTTCCTTCGCAGACCGTAAGCGGAGGCTCCGTGATTGGCTAGGCGGCGAAGACTAGA
CGCTACTCGGGTGGAGGCCTGACCTGAGTAAATGGCGGGTGTCGCAGCAGGTCCACGACTTCTTCGACAGCGTCTTCC
CGTTCTGGCAGTAGGACTGGTAAGTAGTCGGCAGAAGGCTCGACAAACTCGAGAAACTGTTCTAGGAAGACTACCGGC
TCCCGTCCCATCGAAAGAACCCGTGAGGGTCGCTTCGGCAGCTGAAGAAAAGGATCACTCAAGCTACACAAATAATTC
CCATAGATCGTAATGTAATGTAGAGTTGAGGATAGGTCGCACCCACGGGTCACAGGATGGTTGATGTTAGGCCGCCTG
AAAATGCATGTCCACAACCGGCAACACGGGCCTGCCCTCTAGCTCAGGGCACTAGCCTAGCGGTTCTATACGCTGTTA
AAACGATAATCGTTTCATCGGGCCCTATACCTCGTCAACAACCGGTGGTTTTTAAACCTCTTCGGTGACCTCGTCGGC
CTCTTACCCATGTGGATGTTCCGGTGGACCAAGTACGTCAAGGCCCGCCAGGACACCGCTAGGACCGACAGCCACGAG
TTCCTTGGTGAGGAGCATTTTCACGCTGAATAAGTCTGTTGCCACTCACCAAGGTCACCTTTGTTTACTATATTGCGA
ATGTTAAGAACCTTTGTTTAAGCGATCTAAAATCAATCTTAACGGACTAAGGTGTGGGAAGAATCAAAAAAAGTTACT
CTACATATCAAATATCAAAACGTCTTTTATTTATTTAAAGTAAATTGAGCGCTTGTACAACTTCTATACTTATAATTA
CTCTACGCTCATTGTAAAATTAAACGTCTACCAACGGTAGAACTAACCGGAGTAGAAAAACCCGGTTGTTGAGTGCGT
TCACCCGCACTACTTATAGTTGCCTCGGTAGAAGGAGAAGGACTGGTTGTACTGGAAAGTTTTGCAGAAACGGTGCTA
TTTACATTCAGAACAAATCTTATGTAAACGTATAATTATTAAATGATTGAAAGATTACTTAGCTAAGCTAAATCCACA
XmaI
9136
9214
9292
9370
9448
9526
9604
9682
9760
9838
9916
9994
10072
10150
10228
10306
10384
TCACCTCAGAGCTGCCAGTTTTTATGAGGGAGGCCCGAAGTCGACTTTATCGCTGTGACACATACTTTCTGGGCAAAA
CGATTGCCGAATTACCGCTTTTTCTCACAGTGCCACTGGTCTTCACGGCGATTGCCTATCCGATGATCGGACTGCGGG
CCGGAGTGCTGCACTTCTTCAACTGCCTGGCGCTGGTCACTCTGGTGGCCAATGTGTCAACGTCCTTCGGATATCTAA
TATCCTGCGCCAGCTCCTCGACCTCGATGGCGCTGTCTGTGGGTCCGCCGGTTATCATACCATTCCTGCTCTTTGGCG
GCTTCTTCTTGAACTCGGGCTCGGTGCCAGTATACCTCAAATGGTTGTCGTACCTCTCATGGTTCCGTTACGCCAACG
AGGGTCTGCTGATTAACCAATGGGCGGACGTGGAGCCGGGCGAAATTAGCTGCACATCGTCGAACACCACGTGCCCCA
GTTCGGGCAAGGTCATCCTGGAGACGCTTAACTTCTCCGCCGCCGATCTGCCGCTGGACTACGTGGGTCTGGCCATTC
TCATCGTGAGCTTCCGGGTGCTCGCATATCTGGCTCTAAGACTTCGGGCCCGACGCAAGGAGTAGCCGACATATATCC
GAAATAACTGCTTGTTTTTTTTTTTACCATTATTACCATCGTGTTTACTGTTTATTGCCCCCTCAAAAAGCTAATGTA
ATTATATTTGTGCCAATAAAAACAAGATATGACCTATAGAATACAAGTATTTCCCCTTCGAACATCCCCACAAGTAGA
CTTTGGATTTGTCTTCTAACCAAAAGACTTACACACCTGCATACCTTACATCAAAAACTCGTTTATCGCTACATAAAA
CACCGGGATATATTTTTTATATACATACTTTTCAAATCGCGCGCCCTCTTCATAATTCACCTCCACCACACCACGTTT
CGTAGTTGCTCTTTCGCTGTCTCCCACCCGCTCTCCGCAACACATTCACCTTTTGTTCGACGACCTTGGAGCGACTGT
CGTTAGTTCCGCGCGATTCGGTTCGCTCAAATGGTTCCGAGTGGTTCATTTCGTCTCAATAGAAATTAGTAATAAATA
TTTGTATGTACAATTTATTTGCTCCAATATATTTGTATATATTTCCCTCACAGCTATATTTATTCTAATTTAATATTA
TGACTTTTTAAGGTAATTTTTTGTGACCTGTTCGGAGTGATTAGCGTTACAATTTGAACTGAAAGTGACATCCAGTGT
TTGTTCCTTGTGTAGATGCATCTCAAAAAAATGGTGGGCATAATAGTGTTGTTTATATATATCAAAAATAACAACTAT
AGTGGAGTCTCGACGGTCAAAAATACTCCCTCCGGGCTTCAGCTGAAATAGCGACACTGTGTATGAAAGACCCGTTTT
GCTAACGGCTTAATGGCGAAAAAGAGTGTCACGGTGACCAGAAGTGCCGCTAACGGATAGGCTACTAGCCTGACGCCC
GGCCTCACGACGTGAAGAAGTTGACGGACCGCGACCAGTGAGACCACCGGTTACACAGTTGCAGGAAGCCTATAGATT
ATAGGACGCGGTCGAGGAGCTGGAGCTACCGCGACAGACACCCAGGCGGCCAATAGTATGGTAAGGACGAGAAACCGC
CGAAGAAGAACTTGAGCCCGAGCCACGGTCATATGGAGTTTACCAACAGCATGGAGAGTACCAAGGCAATGCGGTTGC
TCCCAGACGACTAATTGGTTACCCGCCTGCACCTCGGCCCGCTTTAATCGACGTGTAGCAGCTTGTGGTGCACGGGGT
CAAGCCCGTTCCAGTAGGACCTCTGCGAATTGAAGAGGCGGCGGCTAGACGGCGACCTGATGCACCCAGACCGGTAAG
AGTAGCACTCGAAGGCCCACGAGCGTATAGACCGAGATTCTGAAGCCCGGGCTGCGTTCCTCATCGGCTGTATATAGG
CTTTATTGACGAACAAAAAAAAAAATGGTAATAATGGTAGCACAAATGACAAATAACGGGGGAGTTTTTCGATTACAT
TAATATAAACACGGTTATTTTTGTTCTATACTGGATATCTTATGTTCATAAAGGGGAAGCTTGTAGGGGTGTTCATCT
GAAACCTAAACAGAAGATTGGTTTTCTGAATGTGTGGACGTATGGAATGTAGTTTTTGAGCAAATAGCGATGTATTTT
GTGGCCCTATATAAAAAATATATGTATGAAAAGTTTAGCGCGCGGGAGAAGTATTAAGTGGAGGTGGTGTGGTGCAAA
GCATCAACGAGAAAGCGACAGAGGGTGGGCGAGAGGCGTTGTGTAAGTGGAAAACAAGCTGCTGGAACCTCGCTGACA
GCAATCAAGGCGCGCTAAGCCAAGCGAGTTTACCAAGGCTCACCAAGTAAAGCAGAGTTATCTTTAATCATTATTTAT
AAACATACATGTTAAATAAACGAGGTTATATAAACATATATAAAGGGAGTGTCGATATAAATAAGATTAAATTATAAT
ACTGAAAAATTCCATTAAAAAACACTGGACAAGCCTCACTAATCGCAATGTTAAACTTGACTTTCACTGTAGGTCACA
AACAAGGAACACATCTACGTAGAGTTTTTTTACCACCCGTATTATCACAACAAATATATATAGTTTTTATTGTTGATA
10462
10540
10618
10696
10774
10852
10930
11008
11086
11164
11242
11320
11398
11476
11554
11632
11710
AATAATAAGAATACATTTAATTTAGAAAATGCTTGGATTTCACTGGAACTAGAATTAATTCGGCTGCTGCTCTAAACG
ACGCATTTCGTACTCCAAAGTACGAATTTTTTCCCTCAAGCTCTTATTTTCATTAAACAATGAACAGGACCTAACGCA
CAGTCACGTTATTGTTTACATAAATGATTTTTTTTACTATTCAAACTTACTCTGTTTGTGTACTCCCACTGGTATAGC
CTTCTTTTATCTTTTCTGGTTCAGGCTCTATCACTTTACTAGGTACGGCATCTGCGTTGAGTCGCCTCCTTTTAAATG
TCTGACCTTTTGCAGGTGCAGCCTTCCACTGCGAATCATTAAAGTGGGTATCACAAATTTGGGAGTTTTCACCAAGGC
TGCACCCAAGGCTCTGCTCCCACAATTTTCTCTTAATAGCACACTTCGGCACGTGAATTAATTTTACTCCAGTCACAG
CTTTGCAGCAAAATTTGCAATATTTCATTTTTTTTTATTCCACGTAAGGGTTAATGTTTTCAAAAAAAAATTCGTCCG
CACACAACCTTTCCTCTCAACAAGCAAACGTGCACTGAATTTAAGTGTATACTTCGGTAAGCTTCGGCTATCGACGGG
ACCACCTTATGTTATTTCATCATG
TTATTATTCTTATGTAAATTAAATCTTTTACGAACCTAAAGTGACCTTGATCTTAATTAAGCCGACGACGAGATTTGC
TGCGTAAAGCATGAGGTTTCATGCTTAAAAAAGGGAGTTCGAGAATAAAAGTAATTTGTTACTTGTCCTGGATTGCGT
GTCAGTGCAATAACAAATGTATTTACTAAAAAAAATGATAAGTTTGAATGAGACAAACACATGAGGGTGACCATATCG
GAAGAAAATAGAAAAGACCAAGTCCGAGATAGTGAAATGATCCATGCCGTAGACGCAACTCAGCGGAGGAAAATTTAC
AGACTGGAAAACGTCCACGTCGGAAGGTGACGCTTAGTAATTTCACCCATAGTGTTTAAACCCTCAAAAGTGGTTCCG
ACGTGGGTTCCGAGACGAGGGTGTTAAAAGAGAATTATCGTGTGAAGCCGTGCACTTAATTAAAATGAGGTCAGTGTC
GAAACGTCGTTTTAAACGTTATAAAGTAAAAAAAAATAAGGTGCATTCCCAATTACAAAAGTTTTTTTTTAAGCAGGC
GTGTGTTGGAAAGGAGAGTTGTTCGTTTGCACGTGACTTAAATTCACATATGAAGCCATTCGAAGCCGATAGCTGCCC
TGGTGGAATACAATAAAGTAGTAC
5' P
5' P
5' P
11788
11866
11944
12022
12100
12178
12256
12334
12412