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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.dna
Map and Sequence File:    Download    Open   
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) AclI (364) BbsI (437) HincII - HpaI (501) AflII (554) AgeI - SgrAI (677) 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
AgeI  (677)
1 site
A C C G G T T G G C C A
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
ORF:  628 .. 1080  =  453 bp
ORF:  150 amino acids  =  17.4 kDa
ORF:  5689 .. 6699  =  1011 bp
ORF:  336 amino acids  =  37.5 kDa
ORF:  641 .. 5305  =  4665 bp
ORF:  1554 amino acids  =  178.6 kDa
ORF:  5076 .. 5309  =  234 bp
ORF:  77 amino acids  =  9.0 kDa
ORF:  5940 .. 6197  =  258 bp
ORF:  85 amino acids  =  9.1 kDa
ORF:  6207 .. 6485  =  279 bp
ORF:  92 amino acids  =  9.4 kDa
ORF:  394 .. 690  =  297 bp
ORF:  98 amino acids  =  10.8 kDa
ORF:  2098 .. 2391  =  294 bp
ORF:  97 amino acids  =  10.8 kDa
ORF:  2662 .. 3111  =  450 bp
ORF:  149 amino acids  =  17.3 kDa
ORF:  4837 .. 5250  =  414 bp
ORF:  137 amino acids  =  14.3 kDa
ORF:  5947 .. 6330  =  384 bp
ORF:  127 amino acids  =  14.6 kDa
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