construct maps
TRANSCRIPT
[email protected] 310.342.5500April 10, 2008
Rapidly discover and develop drugs that prevent or slow HuntingtRapidly discover and develop drugs that prevent or slow Huntington diseaseon disease
Construct MapsConstruct Maps
pcDNA3.1pcDNA3.1Courtesy of Drs. Linda Dong and Janine Steiger (Courtesy of Drs. Linda Dong and Janine Steiger (CombinatoRxCombinatoRx, Inc.), Inc.)
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Htt-Q23, [codon scheme], 1-3144, human
Cloning vector: pcDNA3.1(+), 5428 bpInsert: Full human Htt Q23 with XXXX codon, ~9.5 kbCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAG.Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, RV and PmeI were deleted.Diagnostic check: XhoI digestion should release two bands: 9 kb, 6 kb; Not I digestion should linearize the 15 kb bandSelection: Ampicillin
Q23
Not I
EcoRV/PmeI blunt
hu Htt FLKozak ATGSTOP codon
Xho I
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Htt-Q73, [codon scheme], 1-3144, human
Q73
hu Htt FL
Cloning vector: pcDNA3.1(+), 5428 bpInsert: Full human Htt Q73 with XXX codon, ~9.6 kbCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAG.Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, RV and PmeI were deleted.Diagnostic check: XhoI digestion should release two bands: 9 kb, 6 kb; Not I digestion should linearize the 15 kb band.Selection: Ampicillin
Not I
EcoRV/PmeI blunt
STOP codonKozak ATG
Xho I
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Htt-Q145, [codon scheme], 1-3144, human
Q145
hu Htt FL
Cloning vector: pcDNA3.1(+), 5428 bpInsert: Full human Htt Q145 with XXX codon, ~9.8 kbCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x;Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, RV and PmeI were deleted.Diagnostic check: XhoI digestion should release two bands: 9.2kb, 6kb; Not I digestion should linearize the 15.2kb band.Selection: Ampicillin
Not I
EcoRV/PmeI bluntSTOP codon
Kozak ATGXho I
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Htt-Q23, [codon scheme], 1-90, human
Cloning vector: pcDNA3.1(+), 5428 bpInsert: Human Htt Exon 1 with Q23 XXX codon, ~300 bpCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAGCloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI / Not I digestion should release two bands (5.4kb,0.3kb); StuI digestion should release two bands: 4.4 kb, 1.3 kb.Selection: Ampicillin
hu Htt E1Q23EcoRV/PmeI blunt
Kozak ATG
Not I
STOP codon
Stu I
Stu I
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Htt-Q73, [codon scheme], 1-90, humanCloning vector: pcDNA3.1(+), 5428 bpInsert: Human Htt Exon 1 with Q73 XXX codon, ~450 bpCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAGCloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI / Not I digestion should release two bands (5.4kb,0.45kb); StuI digestion should release two bands: 4.4 kb, 1.5 kb;Selection: Ampicillin
hu Htt E1Q73EcoRV/PmeI blunt
Kozak ATG
Not I
STOP codon
Stu I
Stu I
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Htt-Q103, rdm, 1-90, humanCloning vector: pcDNA3.1(+), 5428 bpInsert: Human Htt Exon 1 with Q103 random (rdm) codon, ~540 bpCodon scheme:; rdm: x/CAG/CAA/x; Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI / Not I digestion should release two bands (5.4kb,0.5kb); StuI digestion should release two bands: 4.4 kb, 1.6 kb Selection: Ampicillin
STOP codonhu Htt E1Q103EcoRV/PmeI blunt
Kozak ATG
Not I
Stu I
Stu I
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Htt-Q145, [codon scheme], 1-90, humanCloning vector: pcDNA3.1(+), 5428 bpInsert: Human Htt Exon 1 with Q145 XXX codon, ~666 bpCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x;Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI / Not I digestion should release two bands (5.4kb,0.66kb); StuI digestion should release two bands: 4.4 kb, 1.7 kb Selection: Ampicillin
hu Htt E1Q145EcoRV/PmeI blunt
Kozak ATG
Not I
STOP codon
Stu I
Stu I
[email protected] 310.342.5500April 10, 2008
Rapidly discover and develop drugs that prevent or slow HuntingtRapidly discover and develop drugs that prevent or slow Huntington diseaseon disease
Construct MapsConstruct Maps
pcDNA3.1pcDNA3.1--mycCmycCCourtesy of Drs. Linda Dong and Janine Steiger (Courtesy of Drs. Linda Dong and Janine Steiger (CombinatoRxCombinatoRx, Inc.), Inc.)
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Htt-Q23-myc, [codon scheme], 1-3144, humanCloning vector: pcDNA3.1(+) myc tagged frame C, ~5.5 kbInsert: Human Htt full-length with Q23 XXXX codon, 9454 bpCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAG.Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: XhoI digestion should release two bands: 8.9 kb, 6 kb; Not I digestion should linearize the 15 kb bandSelection: Ampicillin
Q23
hu Htt FLNot I
EcoRV/PmeI blunt
STOP codonKozak ATGXho I
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Htt-Q73-myc, [codon scheme], 1-3144, humanCloning vector: pcDNA3.1(+) myc tagged frame C, ~5.5 kbInsert: Human Htt full-length with Q73 XXXX codon, ~9.6 kbCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAG.Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: XhoI digestion should release two bands: 8.9 kb, 6 kb; Not I digestion should linearize the 15 kb bandSelection: Ampicillin
Xho I
Q73
hu Htt FLNot I
EcoRV/PmeI blunt
STOP codonKozak ATG
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Htt-Q145-myc, [codon scheme], 1-3144, humanCloning vector: pcDNA3.1(+) myc tagged frame C, ~5.5 kbInsert: Human Htt full-length with Q145 XXXX codon, ~9.8 kbCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAG.Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: XhoI digestion should release two bands: 9.2 kb, 6 kb; Not I digestion should linearize the 15.2 kb bandSelection: Ampicillin
Q145
hu Htt FLNot I
EcoRV/PmeI blunt
STOP codonKozak ATGXho I
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Htt-Q23-myc, [codon scheme], 1-90, humanCloning vector: pcDNA3.1(+) myc tagged frame C, ~5.5 kbInsert: Human Htt Exon 1 with Q23 XXXX codon, ~ 300 bpCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAG.Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI / Not I digestion should release two bands (5.4kb,0.3kb); StuI digestion should release two bands: 4.4 kb, 1.3 kb. Selection: Ampicillin
STOP codonhu Htt E1Not I
EcoRV/PmeI blunt Q23
Kozak ATG
Stu I
Stu I
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Htt-Q73-myc, [codon scheme], 1-90, humanCloning vector: pcDNA3.1(+) myc tagged frame C, ~5.5 kbInsert: Human Htt Exon 1 with Q73 XXXX codon, ~ 450 bpCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAG.Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI / Not I digestion should release two bands (5.4kb,0.45kb); StuI digestion should release two bands: 4.4 kb, 1.5 kbSelection: Ampicillin
STOP codonhu Htt E1Not I
EcoRV/PmeI blunt Q73
Kozak ATG
Stu I
Stu I
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Htt-Q103-myc, rdm 1-90, humanCloning vector: pcDNA3.1(+) myc tagged frame C, ~5.5 kbInsert: Human Htt Exon 1 with Q103 rdm codon, ~ 540 bpCodon scheme: rdm: x/CAG/CAA/x; Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI / Not I digestion should release two bands (5.4kb,0.5kb); StuI digestion should release two bands: 4.4 kb, 1.6 kbSelection: Ampicillin
STOP codonhu Htt E1Not I
EcoRV/PmeI blunt Q103
Kozak ATG
Stu I
Stu I
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Htt-Q145-myc, [codon scheme], 1-90, humanCloning vector: pcDNA3.1(+) myc tagged frame C, ~5.5 kbInsert: Human Htt Exon 1 with Q73 XXXX codon, ~ 666 bpCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAG.Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI / Not I digestion should release two bands (5.4kb,0.66kb); StuI digestion should release two bands: 4.4 kb, 1.7 kbSelection: Ampicillin
STOP codonhu Htt E1Not I
EcoRV/PmeI blunt Q23
Kozak ATG
Stu I
Stu I
[email protected] 310.342.5500April 10, 2008
Rapidly discover and develop drugs that prevent or slow HuntingtRapidly discover and develop drugs that prevent or slow Huntington diseaseon disease
Construct MapsConstruct Maps
pcDNA5/FRTpcDNA5/FRTCourtesy of Drs. Linda Dong and Janine Steiger (Courtesy of Drs. Linda Dong and Janine Steiger (CombinatoRxCombinatoRx, Inc.), Inc.)
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Htt-Q23-FRT, [codon scheme], 1-3144, human
Cloning vector: pcDNA5/FRT, 5070 bpInsert: Full human Htt Q23 with XXXX codon, ~9.5 kbCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAG.Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, RV and PmeI were deleted.Diagnostic check: XhoI digestion should release two bands: 9 kb, 6 kb; Not I digestion should linearize the 15 kb bandSelection: Ampicillin
STOP codon
EcoRV/PmeI blunt Q23
hu Htt FLNot I
Kozak ATGXho I
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Htt-Q73-FRT, [codon scheme], 1-3144, humanCloning vector: pcDNA5/FRT, 5070 bpInsert: Full human Htt Q73 with XXX codon, ~9.6 kbCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAG.Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, RV and PmeI were deleted.Diagnostic check: XhoI digestion should release two bands: 9 kb, 6 kb; Not I digestion should linearize the 15 kb band.Selection: Ampicillin
STOP codon
EcoRV/PmeI blunt Q73
hu Htt FLNot I
Kozak ATGXho I
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Htt-Q145-FRT, [codon scheme], 1-3144, humanCloning vector: pcDNA5/FRT, 5070 bpInsert: Full human Htt Q145 with XXX codon, ~9.8 kbCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x;Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, RV and PmeI were deleted.Diagnostic check: XhoI digestion should release two bands: 9.2kb, 6kb; Not I digestion should linearize the 15.2kb band.Selection: Ampicillin
STOP codon
EcoRV/PmeI blunt Q145
hu Htt FLNot I
Kozak ATGXho I
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Htt-Q23-FRT, [codon scheme], 1-90, humanCloning vector: pcDNA5/FRT, 5070 bpInsert: Human Htt Exon 1 with Q23 XXX codon, ~300 bpCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAGCloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI/NotI digest should release two bands: 5.0kb + 0.3 kb.Selection: Ampicillin
hu Htt E1Q23EcoRV/PmeI blunt
Kozak ATGNot I
STOP codonStu I
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Htt-Q73-FRT, [codon scheme], 1-90, humanCloning vector: pcDNA5/FRT, 5070 bpInsert: Human Htt Exon 1 with Q73 XXX codon, ~450 bpCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x; CAG: pure CAGCloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI/NotI digest should release two bands: 5.0kb + 0.45 kb Selection: Ampicillin
hu Htt E1Q73EcoRV/PmeI blunt
Kozak ATGNot I
STOP codonStu I
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Htt-Q103-FRT, rdm, 1-90, humanCloning vector: pcDNA5/FRT, 5070 bpInsert: Human Htt Exon 1 with Q103 random (rdm) codon, ~540 bpCodon scheme:; rdm: x/CAG/CAA/x; Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI/NotI digest should release two bands: 5.0kb + 0.5 kb Selection: Ampicillin
hu Htt E1Q103EcoRV/PmeI blunt
Kozak ATGNot I
STOP codonStu I
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Htt-Q145-FRT, [codon scheme], 1-90, humanCloning vector: pcDNA5/FRT, 5070 bpInsert: Human Htt Exon 1 with Q145 XXX codon, ~666 bpCodon scheme: mix [CAA-CAG-CAG-CAA-CAG-CAA]n; rdm: x/CAG/CAA/x;Cloning site: fused EcoRV/PmeI at 5’ and Not I at 3’, EcoRV and PmeI were deletedDiagnostic check: BamHI/NotI digest should release two bands: 5.0kb +0.66kbSelection: Ampicillin
hu Htt E1Q145EcoRV/PmeI blunt
Kozak ATGNot I
STOP codonStu I