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pHR-SFFV-dCas9-BFP

Lentiviral CRISPR interference vector for expressing catalytically inactive dCas9 fused to TagBFP.

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pHR-SFFV-dCas9-BFP Sequence and MappHR-SFFV-dCas9-BFP.dna
Map and Sequence File   
Sequence Author:  Qi & Weissman Labs / Addgene #46910
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 HpaI (13,633) AgeI (12,335) AvrII (11,845) SfiI (11,798) FspI (10,230) PvuI (10,084) AfeI (9121) SgrAI (9035) AbsI - PspXI (8403) SbfI (7767) Bsu36I (7290) BamHI (7034) NruI * (833) FseI (1150) AleI (1576) BstEII (2468) AscI (2692) PflFI - Tth111I (2714) MluI (2754) SV40 NLS SV40 NLS pHR-SFFV-dCas9-BFP 13,936 bp
HpaI  (13,633)
1 site
G T T A A C C A A T T G
AgeI  (12,335)
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.
AvrII  (11,845)
1 site
C C T A G G G G A T C C
SfiI  (11,798)
1 site
G G C C N N N N N G G C C C C G G N N N N N C C G G

Efficient cleavage requires at least two copies of the SfiI
recognition sequence.
Sticky ends from different SfiI sites may not be compatible.
FspI  (10,230)
1 site
T G C G C A A C G C G T
PvuI  (10,084)
1 site
C G A T C G G C T A G C
AfeI  (9121)
1 site
A G C G C T T C G C G A
SgrAI  (9035)
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.
AbsI  (8403)
1 site
C C T C G A G G G G A G C T C C
PspXI  (8403)
1 site
V C T C G A G B B G A G C T C V
SbfI  (7767)
1 site
C C T G C A G G G G A C G T C C
Bsu36I  (7290)
1 site
C C T N A G G G G A N T C C

Sticky ends from different Bsu36I sites may not be compatible.
BamHI  (7034)
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.
NruI  (833)
1 site
T C G C G A A G C G C T
* Blocked by Dam methylation.
FseI  (1150)
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.
AleI  (1576)
1 site
C A C N N N N G T G G T G N N N N C A C
BstEII  (2468)
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.
AscI  (2692)
1 site
G G C G C G C C C C G C G C G G
PflFI  (2714)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by PflFI may be hard to ligate.
Sticky ends from different PflFI sites may not be compatible.
Tth111I  (2714)
1 site
G A C N N N G T C C T G N N N C A G

The 1-base overhangs produced by Tth111I may be hard to ligate.
Sticky ends from different Tth111I sites may not be compatible.
MluI  (2754)
1 site
A C G C G T T G C G C A
dCas9
2801 .. 6904  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   2801 .. 2827  =  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
2801 .. 6904  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   2828 .. 2830  =  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
2801 .. 6904  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   2831 .. 5317  =  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
2801 .. 6904  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   5318 .. 5320  =  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
2801 .. 6904  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   5321 .. 6904  =  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
2801 .. 6904  =  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
9666 .. 10,526  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   9666 .. 9734  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9666 .. 10,526  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   9735 .. 10,526  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
9666 .. 10,526  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
TagBFP
7040 .. 7738  =  699 bp
232 amino acids  =  26.2 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
TagBFP
7040 .. 7738  =  699 bp
232 amino acids  =  26.2 kDa
Product: monomeric blue fluorescent protein
mammalian codon-optimized
LTR
1 .. 634  =  634 bp
long terminal repeat (LTR) from HIV-1
LTR
1 .. 634  =  634 bp
long terminal repeat (LTR) from HIV-1
WPRE
7799 .. 8387  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
7799 .. 8387  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
10,697 .. 11,285  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
10,697 .. 11,285  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
SFFV promoter
2319 .. 2726  =  408 bp
spleen focus-forming virus long terminal repeat
(LTR) promoter
SFFV promoter
2319 .. 2726  =  408 bp
spleen focus-forming virus long terminal repeat
(LTR) promoter
SV40 promoter
11,531 .. 11,860  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
11,531 .. 11,860  =  330 bp
SV40 enhancer and early promoter
LTR (ΔU3)
8497 .. 8730  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
LTR (ΔU3)
8497 .. 8730  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
RRE
1303 .. 1535  =  233 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
1303 .. 1535  =  233 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
SV40 poly(A) signal
13,634 .. 13,768  =  135 bp
SV40 polyadenylation signal
SV40 poly(A) signal
13,634 .. 13,768  =  135 bp
SV40 polyadenylation signal
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
681 .. 806  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
2049 .. 2166  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
2049 .. 2166  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
9561 .. 9665  =  105 bp
AmpR promoter
9561 .. 9665  =  105 bp
HA
6908 .. 6934  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
HA
6908 .. 6934  =  27 bp
9 amino acids  =  1.1 kDa
Product: HA (human influenza hemagglutinin)
epitope tag
SV40 NLS
6953 .. 6973  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6953 .. 6973  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6980 .. 7000  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6980 .. 7000  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 ori
11,711 .. 11,846  =  136 bp
SV40 origin of replication
SV40 ori
11,711 .. 11,846  =  136 bp
SV40 origin of replication
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