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Plasmid Files

pAC154-dual-dCas9VP160-sgExpression

CRISPR activation vector for co-expressing a single guide RNA (sgRNA) with catalytically inactive dCas9 fused to the VP160 transcriptional activation domain.

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pAC154-dual-dCas9VP160-sgExpression Sequence and MappAC154-dual-dCas9VP160-sgExpression.dna
Map and Sequence File   
Sequence Author:  Jaenisch Lab / Addgene #48240
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 BbsI (245) BbsI (267) CMV enhancer ATG HA SV40 NLS pAC154-dual-dCas9VP160-sgExpression 8859 bp
BbsI  (245)
2 sites
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.
BbsI  (267)
2 sites
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.
dCas9
1314 .. 5414  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 1:  
   1314 .. 1337  =  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
1314 .. 5414  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 2:  
   1338 .. 1340  =  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
1314 .. 5414  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 3:  
   1341 .. 3827  =  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
1314 .. 5414  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 4:  
   3828 .. 3830  =  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
1314 .. 5414  =  4101 bp
1367 amino acids  =  158.2 kDa
   Segment 5:  
   3831 .. 5414  =  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
1314 .. 5414  =  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
AmpR
7179 .. 8039  =  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
7179 .. 8039  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   7248 .. 8039  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7179 .. 8039  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
8210 .. 8798  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8210 .. 8798  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
6337 .. 6792  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
6337 .. 6792  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
VP160
5472 .. 5855  =  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
5472 .. 5855  =  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
440 .. 725  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
CMV enhancer
440 .. 725  =  286 bp
human cytomegalovirus immediate early enhancer;
contains an 18-bp deletion relative to the standard
CMV enhancer
chicken β-actin promoter
727 .. 1004  =  278 bp
chicken β-actin promoter
727 .. 1004  =  278 bp
U6 promoter
1 .. 241  =  241 bp
RNA polymerase III promoter for human U6 snRNA
U6 promoter
1 .. 241  =  241 bp
RNA polymerase III promoter for human U6 snRNA
hybrid intron
1005 .. 1233  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
hybrid intron
1005 .. 1233  =  229 bp
hybrid between chicken β-actin (CBA) and minute
virus of mice (MMV) introns (Gray et al., 2011)
bGH poly(A) signal
5906 .. 6113  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5906 .. 6113  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
6122 .. 6262  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AAV2 ITR
6122 .. 6262  =  141 bp
inverted terminal repeat of adeno-associated virus
serotype 2
AmpR promoter
7074 .. 7178  =  105 bp
AmpR promoter
7074 .. 7178  =  105 bp
gRNA scaffold
268 .. 343  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
gRNA scaffold
268 .. 343  =  76 bp
guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system
HA
1254 .. 1280  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
1254 .. 1280  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
1284 .. 1304  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
1284 .. 1304  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5418 .. 5438  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5418 .. 5438  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
polIII terminator
344 .. 349  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
344 .. 349  =  6 bp
RNA polymerase III transcription terminator
ATG
1251 .. 1253  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
1251 .. 1253  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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