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Plasmid Files

p3s-Cas9HC

Mammalian cell vector for expressing S. pyogenes Cas9 with a C-terminal HA epitope tag.

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p3s-Cas9HC Sequence and Mapp3s-Cas9HC.dna
Map and Sequence File   
Sequence Author:  Kim Lab / Addgene #43945
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 SalI - SgrDI (7381) SspI (7264) PvuI (6830) AhdI (6460) DrdI (5675) XbaI (5086) NsiI (5085) PaeR7I - PspXI - XhoI (5074) BstZ17I (5040) SV40 NLS BspEI (5011) AgeI (4957) MreI - SgrAI (4549) KflI (4278) ApoI (127) MfeI (161) NruI (208) MluI (228) SpeI (249) CMV enhancer NdeI (484) SnaBI (590) Acc65I (934) KpnI (938) SbfI (1148) AarI (1319) BstAPI (2436) BsrGI (2459) BstEII (2574) PmlI (3690) p3s-Cas9HC 7383 bp
SalI  (7381)
1 site
G T C G A C C A G C T G
SgrDI  (7381)
1 site
C G T C G A C G G C A G C T G C
SspI  (7264)
1 site
A A T A T T T T A T A A
PvuI  (6830)
1 site
C G A T C G G C T A G C
AhdI  (6460)
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.
DrdI  (5675)
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.
XbaI  (5086)
1 site
T C T A G A A G A T C T
NsiI  (5085)
1 site
A T G C A T T A C G T A
PaeR7I  (5074)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5074)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5074)
1 site
C T C G A G G A G C T C
BstZ17I  (5040)
1 site
G T A T A C C A T A T G
BspEI  (5011)
1 site
T C C G G A A G G C C T
AgeI  (4957)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
MreI  (4549)
1 site
C G C C G G C G G C G G C C G C
SgrAI  (4549)
1 site
C R C C G G Y G G Y G G C C R C

Efficient cleavage requires at least two copies of the SgrAI
recognition sequence.
KflI  (4278)
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.
ApoI  (127)
1 site
R A A T T Y Y T T A A R

ApoI is typically used at 50°C, but is 50% active at 37°C.
MfeI  (161)
1 site
C A A T T G G T T A A C
NruI  (208)
1 site
T C G C G A A G C G C T
MluI  (228)
1 site
A C G C G T T G C G C A
SpeI  (249)
1 site
A C T A G T T G A T C A
NdeI  (484)
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.
SnaBI  (590)
1 site
T A C G T A A T G C A T
Acc65I  (934)
1 site
G G T A C C C C A T G G
KpnI  (938)
1 site
G G T A C C C C A T G G
SbfI  (1148)
1 site
C C T G C A G G G G A C G T C C
AarI  (1319)
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.
BstAPI  (2436)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
BsrGI  (2459)
1 site
T G T A C A A C A T G T

BsrGI is typically used at 37°C, but is even more active at 60°C.
BstEII  (2574)
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.
PmlI  (3690)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
Cas9
901 .. 5004  =  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
901 .. 5004  =  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
AmpR
6387 .. 7247  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   6387 .. 7178  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6387 .. 7247  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7179 .. 7247  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
6387 .. 7247  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
5628 .. 6216  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5628 .. 6216  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
235 .. 614  =  380 bp
human cytomegalovirus immediate early enhancer
bGH poly(A) signal
5145 .. 5369  =  225 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5145 .. 5369  =  225 bp
bovine growth hormone polyadenylation signal
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
615 .. 818  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
AmpR promoter
7248 .. 7352  =  105 bp
AmpR promoter
7248 .. 7352  =  105 bp
HA
5041 .. 5067  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
5041 .. 5067  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
5020 .. 5040  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5020 .. 5040  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
863 .. 881  =  19 bp
promoter for bacteriophage T7 RNA polymerase
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