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pST1374-NLS-flag-linker-Cas9

Plasmid for mammalian cell expression of Cas9 with an N-terminal nuclear localization signal followed by a FLAG® tag.

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pST1374-NLS-flag-linker-Cas9.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Huang Lab / Addgene #44758
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AhdI (8304) FspI (8081) SgrDI (7378) MfeI (7215) SpeI (7127) SnaBI (6790) SacI (6566) Eco53kI (6564) BmtI (6485) NheI (6481) NotI (6444) XbaI (6412) PflMI (6377) RsrII (6118) Bsu36I (5864) PaeR7I - XhoI (5176) BbvCI (5120) BamHI (4974) BstZ17I (258) MscI (943) TspMI - XmaI (1050) SmaI (1052) AvrII (1071) StuI (1076) SexAI * (1303) PmeI (2123) AgeI (2149) PspOMI (2206) ApaI (2210) KflI (2937) BstXI (3532) BmgBI (3807) pST1374-NLS-flag-linker-Cas9 9317 bp
AhdI  (8304)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
FspI  (8081)
1 site
T G C G C A A C G C G T
SgrDI  (7378)
1 site
C G T C G A C G G C A G C T G C
MfeI  (7215)
1 site
C A A T T G G T T A A C
SpeI  (7127)
1 site
A C T A G T T G A T C A
SnaBI  (6790)
1 site
T A C G T A A T G C A T
SacI  (6566)
1 site
G A G C T C C T C G A G
Eco53kI  (6564)
1 site
G A G C T C C T C G A G
BmtI  (6485)
1 site
G C T A G C C G A T C G
NheI  (6481)
1 site
G C T A G C C G A T C G
NotI  (6444)
1 site
G C G G C C G C C G C C G G C G
XbaI  (6412)
1 site
T C T A G A A G A T C T
PflMI  (6377)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
RsrII  (6118)
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.
Bsu36I  (5864)
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.
PaeR7I  (5176)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (5176)
1 site
C T C G A G G A G C T C
BbvCI  (5120)
1 site
C C T C A G C G G A G T C G
BamHI  (4974)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
BstZ17I  (258)
1 site
G T A T A C C A T A T G
MscI  (943)
1 site
T G G C C A A C C G G T
TspMI  (1050)
1 site
C C C G G G G G G C C C
XmaI  (1050)
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.
SmaI  (1052)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AvrII  (1071)
1 site
C C T A G G G G A T C C
StuI  (1076)
1 site
A G G C C T T C C G G A
SexAI  (1303)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
PmeI  (2123)
1 site
G T T T A A A C C A A A T T T G
AgeI  (2149)
1 site
A C C G G T T G G C C A
PspOMI  (2206)
1 site
G G G C C C C C C G G G
ApaI  (2210)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
KflI  (2937)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
BstXI  (3532)
1 site
C C A N N N N N N T G G G G