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Plasmid Files

pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP

CRISPR activation vector encoding TagBFP plus catalytically inactive dCas9 fused to 10 copies of an optimized GCN4_v4 peptide.

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pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP Sequence and MappHRdSV40-dCas9-10xGCN4_v4-P2A-BFP.dna
Map and Sequence File   
Sequence Author:  Vale Lab / Addgene #60903
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 SV40 NLS NotI (12,838) BamHI (12,043) SV40 NLS SV40 NLS SV40 NLS ATG pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP 14,551 bp
NotI  (12,838)
1 site
G C G G C C G C C G C C G G C G
BamHI  (12,043)
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.
dCas9
7810 .. 11,913  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   7810 .. 7836  =  27 bp
   9 amino acids  =  1.1 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
7810 .. 11,913  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   7837 .. 7839  =  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
7810 .. 11,913  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   7840 .. 10,326  =  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
7810 .. 11,913  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   10,327 .. 10,329  =  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
7810 .. 11,913  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   10,330 .. 11,913  =  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
7810 .. 11,913  =  4104 bp
1368 amino acids  =  158.3 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
1014 .. 1874  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1014 .. 1082  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1014 .. 1874  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1083 .. 1874  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
1014 .. 1874  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 1:  GCN4_v4  
   12,073 .. 12,129  =  57 bp
   19 amino acids  =  2.3 kDa
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 2:  
   12,130 .. 12,144  =  15 bp
   5 amino acids  =  363.3 Da
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 3:  GCN4_v4  
   12,145 .. 12,201  =  57 bp
   19 amino acids  =  2.3 kDa
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 4:  
   12,202 .. 12,216  =  15 bp
   5 amino acids  =  363.3 Da
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 5:  GCN4_v4  
   12,217 .. 12,273  =  57 bp
   19 amino acids  =  2.3 kDa
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 6:  
   12,274 .. 12,288  =  15 bp
   5 amino acids  =  363.3 Da
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 7:  GCN4_v4  
   12,289 .. 12,345  =  57 bp
   19 amino acids  =  2.3 kDa
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 8:  
   12,346 .. 12,360  =  15 bp
   5 amino acids  =  363.3 Da
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 9:  GCN4_v4  
   12,361 .. 12,417  =  57 bp
   19 amino acids  =  2.3 kDa
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 10:  
   12,418 .. 12,432  =  15 bp
   5 amino acids  =  363.3 Da
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 11:  GCN4_v4  
   12,433 .. 12,489  =  57 bp
   19 amino acids  =  2.3 kDa
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 12:  
   12,490 .. 12,504  =  15 bp
   5 amino acids  =  363.3 Da
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 13:  GCN4_v4  
   12,505 .. 12,561  =  57 bp
   19 amino acids  =  2.3 kDa
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 14:  
   12,562 .. 12,576  =  15 bp
   5 amino acids  =  363.3 Da
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 15:  GCN4_v4  
   12,577 .. 12,633  =  57 bp
   19 amino acids  =  2.3 kDa
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 16:  
   12,634 .. 12,648  =  15 bp
   5 amino acids  =  363.3 Da
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 17:  GCN4_v4  
   12,649 .. 12,705  =  57 bp
   19 amino acids  =  2.3 kDa
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 18:  
   12,706 .. 12,720  =  15 bp
   5 amino acids  =  363.3 Da
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
   Segment 19:  GCN4_v4  
   12,721 .. 12,777  =  57 bp
   19 amino acids  =  2.3 kDa
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
10xGCN4_v4
12,073 .. 12,777  =  705 bp
235 amino acids  =  26.1 kDa
19 segments
Product: 10 tandem copies of the GCN4 peptide
optimized to improve solubility while preserving
binding to an scFv-GFP fusion protein (Tanenbaum
et al., 2014)
TagBFP
12,958 .. 13,656  =  699 bp
233 amino acids  =  26.3 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
TagBFP
12,958 .. 13,656  =  699 bp
233 amino acids  =  26.3 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
LTR
5285 .. 5918  =  634 bp
long terminal repeat (LTR) from HIV-1
LTR
5285 .. 5918  =  634 bp
long terminal repeat (LTR) from HIV-1
ori
2045 .. 2633  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
2045 .. 2633  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
WPRE
13,708 .. 14,296  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
13,708 .. 14,296  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
RRE
6582 .. 6815  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
6582 .. 6815  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
LTR (ΔU3)
14,395 .. 77  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
LTR (ΔU3)
14,395 .. 77  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
SV40 promoter
7509 .. 7719  =  211 bp
SV40 early promoter
SV40 promoter
7509 .. 7719  =  211 bp
SV40 early promoter
SV40 poly(A) signal
4982 .. 5116  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
4982 .. 5116  =  135 bp
SV40 polyadenylation signal
HIV-1 Ψ
5965 .. 6090  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
5965 .. 6090  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
7329 .. 7446  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
7329 .. 7446  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
909 .. 1013  =  105 bp
AmpR promoter
909 .. 1013  =  105 bp
P2A
12,889 .. 12,945  =  57 bp
19 amino acids  =  2.0 kDa
Product: 2A peptide from porcine teschovirus-1
polyprotein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
P2A
12,889 .. 12,945  =  57 bp
19 amino acids  =  2.0 kDa
Product: 2A peptide from porcine teschovirus-1
polyprotein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
HA
11,917 .. 11,943  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
11,917 .. 11,943  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
7735 .. 7755  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
7735 .. 7755  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
11,962 .. 11,982  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
11,962 .. 11,982  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
11,989 .. 12,009  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
11,989 .. 12,009  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
12,859 .. 12,879  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
12,859 .. 12,879  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
13,666 .. 13,686  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
13,666 .. 13,686  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
7732 .. 7734  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
7732 .. 7734  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
7578 .. 7713  =  136 bp
SV40 origin of replication
SV40 ori
7578 .. 7713  =  136 bp
SV40 origin of replication
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