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Plasmid Files

pCS2+hSpCas9

Plasmid for in vitro transcription of human codon-optimized S. pyogenes Cas9 using the SP6 promoter.

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pCS2+hSpCas9.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Kinoshita Lab / Addgene #51815
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HindIII (28) NdeI (7909) BseRI (7601) SalI (7356) BpmI (5982) AflIII - PciI (5019) BspQI - SapI (4903) BssHII (4658) KpnI (4625) Acc65I (4621) NsiI (4612) SacII (4600) NotI (4594) MfeI (4535) HpaI (4526) SnaBI (4388) XbaI (4363) BamHI (79) NcoI (89) BglII (458) PasI (1977) EcoRV (2061) Eco53kI (2445) SacI (2447) PmlI (2997) SphI (3474) KflI (3585) BsaBI * (3706) FseI (4340) pCS2+hSpCas9 8343 bp
HindIII  (28)
1 site
A A G C T T T T C G A A
NdeI  (7909)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional nucleotides.
BseRI  (7601)
1 site
G A G G A G ( N ) 8 N N C T C C T C ( N ) 8

Sticky ends from different BseRI sites may not be compatible.
BseRI quickly loses activity at 37°C.
Prolonged incubation with BseRI may lead to degradation of the DNA.
SalI  (7356)
1 site
G T C G A C C A G C T G
BpmI  (5982)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
AflIII  (5019)
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  (5019)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BspQI  (4903)
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  (4903)
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.
BssHII  (4658)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
KpnI  (4625)
1 site
G G T A C C C C A T G G
Acc65I  (4621)
1 site
G G T A C C C C A T G G
NsiI  (4612)
1 site
A T G C A T T A C G T A
SacII  (4600)
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.
NotI  (4594)
1 site
G C G G C C G C C G C C G G C G
MfeI  (4535)
1 site
C A A T T G G T T A A C
HpaI  (4526)
1 site
G T T A A C C A A T T G
SnaBI  (4388)
1 site
T A C G T A A T G C A T
XbaI  (4363)
1 site
T C T A G A A G A T C T
BamHI  (79)
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.
NcoI  (89)
1 site
C C A T G G G G T A C C
BglII  (458)
1 site
A G A T C T T C T A G A
PasI  (1977)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
EcoRV  (2061)
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.
Eco53kI  (2445)
1 site
G A G C T C C T C G A G
SacI  (2447)
1 site
G A G C T C C T C G A G
PmlI  (2997)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
SphI  (3474)
1 site
G C A T G C C G T A C G
KflI  (3585)
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.
BsaBI  (3706)
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.
FseI  (4340)
1 site
G G C C G G C C C C G G C C G G

FseI gradually loses activity when stored at -20°C.
Cas9
211 .. 4311  =  4101 bp
1367 amino acids  =  158.3 kDa
Product: Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
211 .. 4311  =  4101 bp
1367 amino acids  =  158.3 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
7359 .. 8343  =  985 bp
enhancer/promoter region of simian cytomegalovirus major immediate early transcription unit IE94
CMV IE94 promoter
7359 .. 8343  =  985 bp
enhancer/promoter region of simian cytomegalovirus major immediate early transcription unit IE94
AmpR
5839 .. 6699  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   5839 .. 6630  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5839 .. 6699  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   6631 .. 6699  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
5839 .. 6699  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
5080 .. 5668  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
5080 .. 5668  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
6830 .. 7285  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
6830 .. 7285  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
SV40 poly(A) signal
4392 .. 4526  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
4392 .. 4526  =  135 bp
SV40 polyadenylation signal
AmpR promoter
6700 .. 6804  =  105 bp
AmpR promoter
6700 .. 6804  =  105 bp
3xFLAG
94 .. 159  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags, followed by an enterokinase cleavage site
3xFLAG
94 .. 159  =  66 bp
22 amino acids  =  2.7 kDa
Product: three tandem FLAG® epitope tags, followed by an enterokinase cleavage site
nucleoplasmin NLS
4312 .. 4359  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from nucleoplasmin
nucleoplasmin NLS
4312 .. 4359  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from nucleoplasmin
SV40 NLS
166 .. 186  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
166 .. 186  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SP6 promoter
35 .. 53  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
35 .. 53  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
ATG
91 .. 93  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
91 .. 93  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ORF:  91 .. 4362  =  4272 bp
ORF:  1423 amino acids  =  164.6 kDa
ORF:  5969 .. 6235  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  7394 .. 7792  =  399 bp
ORF:  132 amino acids  =  14.3 kDa
ORF:  7377 .. 7625  =  249 bp
ORF:  82 amino acids  =  9.1 kDa
ORF:  8025 .. 8297  =  273 bp
ORF:  90 amino acids  =  9.5 kDa
ORF:  61 .. 2727  =  2667 bp
ORF:  888 amino acids  =  93.5 kDa
ORF:  5839 .. 6699  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  1557 .. 1850  =  294 bp
ORF:  97 amino acids  =  10.8 kDa
ORF:  2121 .. 2570  =  450 bp
ORF:  149 amino acids  =  17.3 kDa
ORF:  7068 .. 7364  =  297 bp
ORF:  98 amino acids  =  10.6 kDa
ORF:  7298 .. 7654  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  7997 .. 8278  =  282 bp
ORF:  93 amino acids  =  10.1 kDa
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