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Plasmid Files

pAC94-pmax-dCas9VP160-2A-puro

CRISPR activation vector for expressing a puromycin resistance marker plus catalytically inactive dCas9 fused to the VP160 transcriptional activation domain.

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pAC94-pmax-dCas9VP160-2A-puro Sequence and MappAC94-pmax-dCas9VP160-2A-puro.dna
Map and Sequence File   
Sequence Author:  Jaenisch Lab / Addgene #48226
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 NsiI (8280) FspI (7995) ApaLI (7017) PciI (6703) AflII (6658) HpaI (6566) PsiI (6546) Bts α I (6492) MluI (6428) BstBI (6384) XbaI (6367) PspXI (6361) NotI (6355) PacI (6301) SexAI * (6264) BpmI (6195) BstEII (5831) BsiWI (5753) AvrII (5685) BspDI - ClaI (5614) AscI (5219) BamHI (5211) FseI (5208) KflI (4442) BspHI (4189) CMV enhancer NdeI (387) SnaBI (493) attB1 SgrAI (993) ATG HA SV40 NLS AhdI (3083) Eco53kI (3302) SacI (3304) pAC94-pmax-dCas9VP160-2A-puro 8282 bp
NsiI  (8280)
1 site
A T G C A T T A C G T A
FspI  (7995)
1 site
T G C G C A A C G C G T
ApaLI  (7017)
1 site
G T G C A C C A C G T G
PciI  (6703)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
AflII  (6658)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
HpaI  (6566)
1 site
G T T A A C C A A T T G
PsiI  (6546)
1 site
T T A T A A A A T A T T
BtsαI  (6492)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsI sites may not be compatible.
MluI  (6428)
1 site
A C G C G T T G C G C A
BstBI  (6384)
1 site
T T C G A A A A G C T T
XbaI  (6367)
1 site
T C T A G A A G A T C T
PspXI  (6361)
1 site
V C T C G A G B B G A G C T C V
NotI  (6355)
1 site
G C G G C C G C C G C C G G C G
PacI  (6301)
1 site
T T A A T T A A A A T T A A T T
SexAI  (6264)
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.
BpmI  (6195)
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.
BstEII  (5831)
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.
BsiWI  (5753)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
AvrII  (5685)
1 site
C C T A G G G G A T C C
BspDI  (5614)
1 site
A T C G A T T A G C T A
ClaI  (5614)
1 site
A T C G A T T A G C T A
AscI  (5219)
1 site
G G C G C G C C C C G C G C G G
BamHI  (5211)
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.
FseI  (5208)
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.
KflI  (4442)
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.
BspHI  (4189)
1 site
T C A T G A A G T A C T
NdeI  (387)
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  (493)
1 site
T A C G T A A T G C A T
SgrAI  (993)
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.
AhdI  (3083)
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.
Eco53kI  (3302)
1 site
G A G C T C C T C G A G
SacI  (3304)
1 site
G A G C T C C T C G A G
dCas9
1068 .. 5168  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 1:  
   1068 .. 1091  =  24 bp
   8 amino acids  =  923.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
1068 .. 5168  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 2:  
   1092 .. 1094  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
1068 .. 5168  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 3:  
   1095 .. 3581  =  2487 bp
   829 amino acids  =  96.3 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
1068 .. 5168  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 4:  
   3582 .. 3584  =  3 bp
   1 amino acid  =  89.1 Da
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
1068 .. 5168  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 5:  
   3585 .. 5168  =  1584 bp
   528 amino acids  =  60.9 kDa
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
dCas9
1068 .. 5168  =  4101 bp
1367 amino acids  =  158.2 kDa
5 segments
Product: catalytically dead mutant of the Cas9
endonuclease from the Streptococcus pyogenes
Type II CRISPR/Cas system
RNA-guided DNA-binding protein that lacks
endonuclease activity due to the D10A mutation in
the RuvC catalytic domain and the H840A mutation
in the HNH catalytic domain
NeoR/KanR
7432 .. 8226  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
7432 .. 8226  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
PuroR
5697 .. 6296  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
5697 .. 6296  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
6764 .. 7351  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6764 .. 7351  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
VP160
5226 .. 5609  =  384 bp
128 amino acids  =  14.0 kDa
Product: 10 tandem repeats of the minimal
activation domain of herpes simplex virus VP16
(Cheng et al., 2013)
VP160
5226 .. 5609  =  384 bp
128 amino acids  =  14.0 kDa
Product: 10 tandem repeats of the minimal
activation domain of herpes simplex virus VP16
(Cheng et al., 2013)
CMV enhancer
138 .. 517  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
138 .. 517  =  380 bp
human cytomegalovirus immediate early enhancer
CMV promoter
518 .. 721  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
518 .. 721  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
chimeric intron
813 .. 945  =  133 bp
chimera between introns from human β-globin and
immunoglobulin heavy chain genes
chimeric intron
813 .. 945  =  133 bp
chimera between introns from human β-globin and
immunoglobulin heavy chain genes
SV40 poly(A) signal
6485 .. 6566  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
6485 .. 6566  =  82 bp
SV40 polyadenylation signal
T2A
5634 .. 5687  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
T2A
5634 .. 5687  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
HA
1008 .. 1034  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
1008 .. 1034  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
attB1
960 .. 984  =  25 bp
recombination site for the Gateway® BP reaction
attB1
960 .. 984  =  25 bp
recombination site for the Gateway® BP reaction
attB2
6312 .. 6336  =  25 bp
recombination site for the Gateway® BP reaction
attB2
6312 .. 6336  =  25 bp
recombination site for the Gateway® BP reaction
SV40 NLS
1038 .. 1058  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1038 .. 1058  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5172 .. 5192  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5172 .. 5192  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
1005 .. 1007  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1005 .. 1007  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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