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pMSCV-LTR-dCas9-p65AD-BFP

Retroviral CRISPR activation vector for expressing TagBFP plus catalytically inactive dCas9 fused to the RelA (p65) transcriptional activation domain.

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pMSCV-LTR-dCas9-p65AD-BFP Sequence and MappMSCV-LTR-dCas9-p65AD-BFP.dna
Map and Sequence File   
Sequence Author:  Qi & Weissman Labs / Addgene #46913
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 SgrAI (11,809) NdeI (11,437) NsiI (8303) SacII (7864) RsrII (7766) BsiWI (7706) BfuAI - BspMI (7639) AgeI (7240) EcoRI (7120) PaeR7I - XhoI (7108) BamHI (7096) PshAI (7024) BbsI (6692) BglII (1410) NcoI (1421) PflMI (1722) SgrDI (1789) BsaBI * (2240) PsiI (2540) MluI (5256) SV40 NLS SV40 NLS pMSCV-LTR-dCas9-p65AD-BFP 11,987 bp
SgrAI  (11,809)
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.
NdeI  (11,437)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
NsiI  (8303)
1 site
A T G C A T T A C G T A
SacII  (7864)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
RsrII  (7766)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
BsiWI  (7706)
1 site
C G T A C G G C A T G C

BsiWI is typically used at 55°C, but is 50% active at 37°C.
BfuAI  (7639)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (7639)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
AgeI  (7240)
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.
EcoRI  (7120)
1 site
G A A T T C C T T A A G
PaeR7I  (7108)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (7108)
1 site
C T C G A G G A G C T C
BamHI  (7096)
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.
PshAI  (7024)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
BbsI  (6692)
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.
BglII  (1410)
1 site
A G A T C T T C T A G A
NcoI  (1421)
1 site
C C A T G G G G T A C C
PflMI  (1722)
1 site
C C A N N N N N T G G G G T N N N N N A C C

Sticky ends from different PflMI sites may not be compatible.
SgrDI  (1789)
1 site
C G T C G A C G G C A G C T G C
BsaBI  (2240)
1 site
G A T N N N N A T C C T A N N N N T A G
* Blocked by Dam methylation.
PsiI  (2540)
1 site
T T A T A A A A T A T T
MluI  (5256)
1 site
A C G C G T T G C G C A
dCas9
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   1423 .. 1449  =  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
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   1450 .. 1452  =  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
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   1453 .. 3939  =  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
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   3940 .. 3942  =  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
1423 .. 5526  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   3943 .. 5526  =  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
1423 .. 5526  =  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
10,193 .. 11,053  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   10,193 .. 10,984  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,193 .. 11,053  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   10,985 .. 11,053  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
10,193 .. 11,053  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TagBFP
6400 .. 7095  =  696 bp
232 amino acids  =  26.2 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
TagBFP
6400 .. 7095  =  696 bp
232 amino acids  =  26.2 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
PuroR
7650 .. 8249  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
7650 .. 8249  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
ori
9434 .. 10,022  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9434 .. 10,022  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
5' LTR
11,986 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem
cell virus
5' LTR
11,986 .. 515  =  517 bp
5' long terminal repeat from murine embryonic stem
cell virus
3' LTR
8349 .. 8863  =  515 bp
3' long terminal repeat from murine embryonic stem
cell virus
3' LTR
8349 .. 8863  =  515 bp
3' long terminal repeat from murine embryonic stem
cell virus
PGK promoter
7130 .. 7629  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
7130 .. 7629  =  500 bp
mouse phosphoglycerate kinase 1 promoter
gag (truncated)
987 .. 1403  =  417 bp
truncated MMLV gag gene lacking the start codon
gag (truncated)
987 .. 1403  =  417 bp
truncated MMLV gag gene lacking the start codon
RelA (p65) AD
6022 .. 6393  =  372 bp
124 amino acids  =  13.0 kDa
Product: transcriptional activation domain of human
RelA, also known as p65 (O'Shea and Perkins, 2008)
RelA (p65) AD
6022 .. 6393  =  372 bp
124 amino acids  =  13.0 kDa
Product: transcriptional activation domain of human
RelA, also known as p65 (O'Shea and Perkins, 2008)
MESV Ψ
579 .. 920  =  342 bp
packaging signal of murine embryonic stem cell
virus
MESV Ψ
579 .. 920  =  342 bp
packaging signal of murine embryonic stem cell
virus
AmpR promoter
11,054 .. 11,158  =  105 bp
AmpR promoter
11,054 .. 11,158  =  105 bp
SV40 NLS
5536 .. 5556  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5536 .. 5556  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5572 .. 5592  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
5572 .. 5592  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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