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Plasmid Files

pCS2-Cas9

S. pyogenes Cas9 plasmid for in vivo expression or in vitro mRNA production.

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pCS2-Cas9.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Schier Lab / Addgene #47322
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BspQI - SapI (8207) AhdI (7202) DraIII (6062) BspDI - ClaI (4684) NcoI (4673) EcoRV (4646) BsmBI (4509) SbfI (4432) XcmI (4325) Acc65I (273) KpnI (277) PspOMI (279) ApaI (283) NsiI (290) NotI - SacII (300) MfeI (359) HpaI (372) SnaBI (510) XbaI (531) AscI (716) EcoRI (849) PaeR7I - XhoI (1128) AarI (1411) NruI (1435) FspAI (1725) PmlI (1886) BmgBI (2165) Eco53kI (3107) SacI (3109) TspMI - XmaI (3305) SmaI (3307) Bsu36I (3406) BlpI (3724) pCS2-Cas9 8215 bp
BspQI  (8207)
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  (8207)
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.
AhdI  (7202)
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.
DraIII  (6062)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
BspDI  (4684)
1 site
A T C G A T T A G C T A
ClaI  (4684)
1 site
A T C G A T T A G C T A
NcoI  (4673)
1 site
C C A T G G G G T A C C
EcoRV  (4646)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
BsmBI  (4509)
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.
SbfI  (4432)
1 site
C C T G C A G G G G A C G T C C
XcmI  (4325)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
Acc65I  (273)
1 site
G G T A C C C C A T G G
KpnI  (277)
1 site
G G T A C C C C A T G G
PspOMI  (279)
1 site
G G G C C C C C C G G G
ApaI  (283)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
NsiI  (290)
1 site
A T G C A T T A C G T A
NotI  (300)
1 site
G C G G C C G C C G C C G G C G
SacII  (300)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII recognition sequence.
MfeI  (359)
1 site
C A A T T G G T T A A C
HpaI  (372)
1 site
G T T A A C C A A T T G
SnaBI  (510)
1 site
T A C G T A A T G C A T
XbaI  (531)
1 site
T C T A G A A G A T C T
AscI  (716)
1 site
G G C G C G C C C C G C G C G G
EcoRI  (849)
1 site
G A A T T C C T T A A G
PaeR7I  (1128)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (1128)
1 site
C T C G A G G A G C T C
AarI  (1411)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
NruI  (1435)
1 site
T C G C G A A G C G C T
FspAI  (1725)
1 site
R T G C G C A Y Y A C G C G T R
PmlI  (1886)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BmgBI  (2165)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
Eco53kI  (3107)
1 site
G A G C T C C T C G A G
SacI  (3109)
1 site
G A G C T C C T C G A G
TspMI  (3305)
1 site
C C C G G G G G G C C C
XmaI  (3305)
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  (3307)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
Bsu36I  (3406)
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.
BlpI  (3724)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
Cas9
573 .. 4676  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
573 .. 4676  =  4104 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
CMV IE94 promoter
4774 .. 5751  =  978 bp
enhancer/promoter region of simian cytomegalovirus major immediate early transcription unit IE94
CMV IE94 promoter
4774 .. 5751  =  978 bp
enhancer/promoter region of simian cytomegalovirus major immediate early transcription unit IE94
AmpR
6415 .. 7275  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6415 .. 6483  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6415 .. 7275  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6484 .. 7275  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6415 .. 7275  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
7446 .. 8034  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
7446 .. 8034  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
5829 .. 6284  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
5829 .. 6284  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
SV40 poly(A) signal
373 .. 507  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
373 .. 507  =  135 bp
SV40 polyadenylation signal
AmpR promoter
6310 .. 6414  =  105 bp
AmpR promoter
6310 .. 6414  =  105 bp
SV40 NLS
540 .. 560  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
540 .. 560  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SP6 promoter
4721 .. 4739  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
4721 .. 4739  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
ORF:  6415 .. 7275  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  630 .. 1163  =  534 bp
ORF:  177 amino acids  =  20.0 kDa
ORF:  2157 .. 2690  =  534 bp
ORF:  177 amino acids  =  19.9 kDa
ORF:  2754 .. 3272  =  519 bp
ORF:  172 amino acids  =  20.1 kDa
ORF:  4197 .. 4727  =  531 bp
ORF:  176 amino acids  =  19.5 kDa
ORF:  4839 .. 5120  =  282 bp
ORF:  93 amino acids  =  10.1 kDa
ORF:  5463 .. 5816  =  354 bp
ORF:  117 amino acids  =  12.9 kDa
ORF:  4820 .. 5092  =  273 bp
ORF:  90 amino acids  =  9.5 kDa
ORF:  5492 .. 5740  =  249 bp
ORF:  82 amino acids  =  9.1 kDa
ORF:  537 .. 4676  =  4140 bp
ORF:  1379 amino acids  =  159.7 kDa
ORF:  5325 .. 5849  =  525 bp
ORF:  174 amino acids  =  19.1 kDa
ORF:  6879 .. 7145  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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