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pCLIP-Cas9-Nuclease-hCMV-Puro

Lentiviral CRISPR vector encoding Cas9 and a puromycin resistance marker expressed from the human CMV promoter.

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pCLIP-Cas9-Nuclease-hCMV-Puro.dna
Map and Sequence File:    Download    Open   
Sequence Author:  transOMIC
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SspI (10,941) PvuI (10,507) FspI (10,359) DrdI (9355) AflIII - PciI (9247) SmaI (9227) TspMI - XmaI (9225) AvrII (9204) PmeI (8879) PsiI (8829) KpnI (8494) Acc65I (8490) Bsu36I (8486) SalI (8366) BstEII (7278) RsrII (7260) BsiWI (7200) PflFI - Tth111I (7186) BsaBI * (6403) PmlI (5694) NheI (405) BmtI (409) NruI * (867) BspQI - SapI (1136) MfeI (1219) BbvCI (1454) AleI (1607) BsmBI (2766) PspXI (2882) XbaI (2888) AfeI (2895) PspOMI (4260) ApaI (4264) pCLIP-Cas9-Nuclease-hCMV-Puro 11,059 bp
SspI  (10,941)
1 site
A A T A T T T T A T A A
PvuI  (10,507)
1 site
C G A T C G G C T A G C
FspI  (10,359)
1 site
T G C G C A A C G C G T
DrdI  (9355)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
AflIII  (9247)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (9247)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
SmaI  (9227)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (9225)
1 site
C C C G G G G G G C C C
XmaI  (9225)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI recognition sequence.
Full cleavage with XmaI may require a long incubation.
AvrII  (9204)
1 site
C C T A G G G G A T C C
PmeI  (8879)
1 site
G T T T A A A C C A A A T T T G
PsiI  (8829)
1 site
T T A T A A A A T A T T
KpnI  (8494)
1 site
G G T A C C C C A T G G
Acc65I  (8490)
1 site
G G T A C C C C A T G G
Bsu36I  (8486)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
SalI  (8366)
1 site
G T C G A C C A G C T G
BstEII  (7278)
1 site
G G T N A C C C C A N T G G

Sticky ends from different BstEII sites may not be compatible.
BstEII is typically used at 60°C, but is 50% active at 37°C.
RsrII  (7260)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BsiWI  (7200)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
PflFI  (7186)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (7186)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
BsaBI  (6403)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
PmlI  (5694)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
NheI  (405)
1 site
G C T A G C C G A T C G
BmtI  (409)
1 site
G C T A G C C G A T C G
NruI  (867)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
BspQI  (1136)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (1136)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
MfeI  (1219)
1 site
C A A T T G G T T A A C
BbvCI  (1454)
1 site
C C T C A G C G G A G T C G
AleI  (1607)
1 site
C A C N N N N G T G G T G N N N N C A C
BsmBI  (2766)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
PspXI  (2882)
1 site
V C T C G A G B B G A G C T C V
XbaI  (2888)
1 site
T C T A G A A G A T C T
AfeI  (2895)
1 site
A G C G C T T C G