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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) ATG SV40 NLS 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
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