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Plasmid Files

pAC149-pCR8-dCas9VP160

Gateway® entry vector with a cassette encoding catalytically inactive dCas9 fused to the VP160 transcriptional activation domain.

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pAC149-pCR8-dCas9VP160 Sequence and MappAC149-pCR8-dCas9VP160.dna
Map and Sequence File   
Sequence Author:  Jaenisch Lab / Addgene #48221
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 PciI (7436) DrdI (7334) BciVI (7238) PaeR7I - PspXI - XhoI (6702) PvuI (6287) BpmI (6158) BstEII (6066) XbaI (5549) BseRI (5536) PacI (5310) BspDI - ClaI (5298) AscI (4903) BamHI (4895) FseI (4892) BsmI (4389) KflI (4126) SphI (4015) BspQI - SapI (111) rrnB T2 terminator AclI (364) BbsI (437) HincII - HpaI (501) AflII (554) AgeI - SgrAI (677) ATG SV40 NLS BglII (999) PasI (2518) DraI (2860) Eco53kI (2986) SacI (2988) PmlI (3538) pAC149-pCR8-dCas9VP160 7442 bp
PciI  (7436)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (7334)
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.
BciVI  (7238)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
PaeR7I  (6702)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (6702)
1 site
V C T C G A G B B G A G C T C V
XhoI  (6702)
1 site
C T C G A G G A G C T C
PvuI  (6287)
1 site
C G A T C G G C T A G C
BpmI  (6158)
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  (6066)
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.
XbaI  (5549)
1 site
T C T A G A A G A T C T
BseRI  (5536)
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.
PacI  (5310)
1 site
T T A A T T A A A A T T A A T T
BspDI  (5298)
1 site
A T C G A T T A G C T A
ClaI  (5298)
1 site
A T C G A T T A G C T A
AscI  (4903)
1 site
G G C G C G C C C C G C G C G G
BamHI  (4895)
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  (4892)
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.
BsmI  (4389)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
KflI  (4126)
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.
SphI  (4015)
1 site
G C A T G C C G T A C G
BspQI  (111)
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  (111)
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.
AclI  (364)
1 site
A A C G T T T T G C A A
BbsI  (437)
1 site
G A A G A C N N C T T C T G N N ( N ) 4

Sticky ends from different BbsI sites may not be compatible.
BbsI gradually loses activity when stored at -20°C.
HincII  (501)
1 site
G T Y R A C C A R Y T G
HpaI  (501)
1 site
G T T A A C C A A T T G
AflII  (554)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
AgeI  (677)
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.
SgrAI  (677)
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  (999)
1 site
A G A T C T T C T A G A
PasI  (2518)
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.
DraI  (2860)
1 site
T T T A A A A A A T T T
Eco53kI  (2986)
1 site
G A G C T C C T C G A G
SacI  (2988)
1 site
G A G C T C C T C G A G
PmlI  (3538)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
dCas9
752 .. 4852  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 1:  
   752 .. 775  =  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
752 .. 4852  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 2:  
   776 .. 778  =  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
752 .. 4852  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 3:  
   779 .. 3265  =  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
752 .. 4852  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 4:  
   3266 .. 3268  =  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
752 .. 4852  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 5:  
   3269 .. 4852  =  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
752 .. 4852  =  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
SmR
5908 .. 6699  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
SmR
5908 .. 6699  =  792 bp
263 amino acids  =  29.3 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
ori
6792 .. 7380  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6792 .. 7380  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
VP160
4910 .. 5293  =  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
4910 .. 5293  =  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)
attL1
569 .. 668  =  100 bp
recombination site for the Gateway® LR reaction
attL1
569 .. 668  =  100 bp
recombination site for the Gateway® LR reaction
attL2
5321 .. 5420  =  100 bp
recombination site for the Gateway® LR reaction
attL2
5321 .. 5420  =  100 bp
recombination site for the Gateway® LR reaction
rrnB T1 terminator
387 .. 473  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
387 .. 473  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T2 terminator
268 .. 295  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
rrnB T2 terminator
268 .. 295  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
HA
692 .. 718  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
692 .. 718  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
722 .. 742  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
722 .. 742  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4856 .. 4876  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4856 .. 4876  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
689 .. 691  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
689 .. 691  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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