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pAC93-pmax-dCas9VP160

CRISPR activation vector for expressing catalytically inactive dCas9 fused to the VP160 transcriptional activation domain.

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pAC93-pmax-dCas9VP160 Sequence and MappAC93-pmax-dCas9VP160.dna
Map and Sequence File   
Sequence Author:  Jaenisch Lab / Addgene #48225
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 NsiI (7605) PluTI (7423) SfoI (7421) NarI (7420) KasI (7419) FspI (7320) PflFI - Tth111I (7303) RsrII (6903) ApaLI (6342) PciI (6028) StuI (6018) AflII (5983) HpaI (5891) PsiI (5871) Bts α I (5817) MluI (5753) BstBI (5709) SacII (5705) XbaI (5692) PaeR7I - PspXI - XhoI (5686) NotI (5680) PacI (5626) BspDI - ClaI (5614) AscI - BssHII (5219) BamHI (5211) FseI (5208) KflI (4442) BspHI (4189) CMV enhancer NdeI (387) SnaBI (493) attB1 SgrAI (993) ATG HA SV40 NLS BglII (1315) PasI (2834) AhdI (3083) Eco53kI (3302) SacI (3304) DraIII (3464) pAC93-pmax-dCas9VP160 7607 bp
NsiI  (7605)
1 site
A T G C A T T A C G T A
PluTI  (7423)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI
recognition sequence.
SfoI  (7421)
1 site
G G C G C C C C G C G G
NarI  (7420)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI
recognition sequence.
KasI  (7419)
1 site
G G C G C C C C G C G G
FspI  (7320)
1 site
T G C G C A A C G C G T
PflFI  (7303)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (7303)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
RsrII  (6903)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
ApaLI  (6342)
1 site
G T G C A C C A C G T G
PciI  (6028)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
StuI  (6018)
1 site
A G G C C T T C C G G A
AflII  (5983)
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  (5891)
1 site
G T T A A C C A A T T G
PsiI  (5871)
1 site
T T A T A A A A T A T T
BtsαI  (5817)
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  (5753)
1 site
A C G C G T T G C G C A
BstBI  (5709)
1 site
T T C G A A A A G C T T
SacII  (5705)
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.
XbaI  (5692)
1 site
T C T A G A A G A T C T
PaeR7I  (5686)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5686)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5686)
1 site
C T C G A G G A G C T C
NotI  (5680)
1 site
G C G G C C G C C G C C G G C G
PacI  (5626)
1 site
T T A A T T A A A A T T A A T T
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
BssHII  (5219)
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.
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.
BglII  (1315)
1 site
A G A T C T T C T A G A
PasI  (2834)
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.
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
DraIII  (3464)
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.
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
6757 .. 7551  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
6757 .. 7551  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
6089 .. 6676  =  588 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6089 .. 6676  =  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
5810 .. 5891  =  82 bp
SV40 polyadenylation signal
SV40 poly(A) signal
5810 .. 5891  =  82 bp
SV40 polyadenylation signal
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
5637 .. 5661  =  25 bp
recombination site for the Gateway® BP reaction
attB2
5637 .. 5661  =  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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