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pHR-SFFV-dCas9-BFP-KRAB

Lentiviral CRISPR interference vector for expressing catalytically inactive dCas9 fused to TagBFP and a KRAB domain.

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pHR-SFFV-dCas9-BFP-KRAB.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Qi & Weissman Labs / Addgene #46911
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HpaI (13,864) AgeI (12,566) AvrII (12,076) SfiI (12,029) FspI (10,461) PvuI (10,315) AfeI (9352) SgrAI (9266) AbsI - PspXI (8634) SbfI (7998) Bsu36I (7290) NruI * (833) FseI (1150) AleI (1576) AscI (2692) PflFI - Tth111I (2714) MluI (2754) BamHI (7034) pHR-SFFV-dCas9-BFP-KRAB 14,167 bp
HpaI  (13,864)
1 site
G T T A A C C A A T T G
AgeI  (12,566)
1 site
A C C G G T T G G C C A
AvrII  (12,076)
1 site
C C T A G G G G A T C C
SfiI  (12,029)
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  (10,461)
1 site
T G C G C A A C G C G T
PvuI  (10,315)
1 site
C G A T C G G C T A G C
AfeI  (9352)
1 site
A G C G C T T C G C G A
SgrAI  (9266)
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  (8634)
1 site
C C T C G A G G G G A G C T C C
PspXI  (8634)
1 site
V C T C G A G B B G A G C T C V
SbfI  (7998)
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.
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
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
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.
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