pAC152-dual-dCas9VP64-sgExpression

CRISPR activation vector for co-expressing a single guide RNA (sgRNA) with catalytically inactive dCas9 fused to the VP64 transcriptional activation domain.

Sequence Author: Jaenisch Lab / Addgene #48238

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BbsI (245) AAV2 ITR BbsI (267) CMV enhancer chicken β-actin promoter ATG SV40 NLS pAC152-dual-dCas9VP64-sgExpression 8625 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.
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
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
dCas9
1314 .. 5414  =  4101 bp
1367 amino acids  =  158.2 kDa
5 segments
   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
5 segments
   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
5 segments
   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
5 segments
   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
5 segments
   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
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
VP64
5472 .. 5621  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)
VP64
5472 .. 5621  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)
AmpR
6945 .. 7805  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 1:  signal sequence  
   6945 .. 7013  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6945 .. 7805  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
   Segment 2:  
   7014 .. 7805  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
6945 .. 7805  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
7976 .. 8564  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
7976 .. 8564  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
6103 .. 6558  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
6103 .. 6558  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
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
5672 .. 5879  =  208 bp
bovine growth hormone polyadenylation signal
bGH poly(A) signal
5672 .. 5879  =  208 bp
bovine growth hormone polyadenylation signal
AAV2 ITR
5888 .. 6028  =  141 bp
inverted terminal repeat of adeno-associated virus serotype 2
AAV2 ITR
5888 .. 6028  =  141 bp
inverted terminal repeat of adeno-associated virus serotype 2
AmpR promoter
6840 .. 6944  =  105 bp
AmpR promoter
6840 .. 6944  =  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
polIII terminator
344 .. 349  =  6 bp
RNA polymerase III transcription terminator
polIII terminator
344 .. 349  =  6 bp
RNA polymerase III transcription terminator
ORF:  5902 .. 6126  =  225 bp
ORF:  74 amino acids  =  8.3 kDa
ORF:  635 .. 982  =  348 bp
ORF:  115 amino acids  =  12.1 kDa
ORF:  825 .. 1136  =  312 bp
ORF:  103 amino acids  =  10.7 kDa
ORF:  1251 .. 5633  =  4383 bp
ORF:  1460 amino acids  =  168.4 kDa
ORF:  6945 .. 7805  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  5802 .. 6083  =  282 bp
ORF:  93 amino acids  =  10.4 kDa
ORF:  2660 .. 2953  =  294 bp
ORF:  97 amino acids  =  10.8 kDa
ORF:  3224 .. 3673  =  450 bp
ORF:  149 amino acids  =  17.3 kDa
ORF:  7409 .. 7675  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
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