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pHAGE EF1α dCas9-VP64

Lentiviral CRISPR activation vector for constitutive expression of catalytically inactive dCas9 fused to the VP64 transcriptional activation domain.

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pHAGE EF1-alpha dCas9-VP64.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Maehr & Wolfe Labs / Addgene #50918
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BamHI (12,567) BstZ17I (12,476) SgrAI (12,447) AscI (12,295) AarI (11,600) FspAI (11,290) SalI (8880) XcmI (8691) SbfI (8587) NcoI (8338) AleI (6449) PspXI (39) SphI (165) BsiWI (702) RsrII (762) BstEII (780) SexAI * (1213) TspMI - XmaI (2230) SmaI (2232) AhdI (3255) SfiI (4375) AvrII (4422) pHAGE EF1 α dCas9-VP64 12,794 bp
BamHI  (12,567)
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.
BstZ17I  (12,476)
1 site
G T A T A C C A T A T G
SgrAI  (12,447)
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.
AscI  (12,295)
1 site
G G C G C G C C C C G C G C G G
AarI  (11,600)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
FspAI  (11,290)
1 site
R T G C G C A Y Y A C G C G T R
SalI  (8880)
1 site
G T C G A C C A G C T G
XcmI  (8691)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
SbfI  (8587)
1 site
C C T G C A G G G G A C G T C C
NcoI  (8338)
1 site
C C A T G G G G T A C C
AleI  (6449)
1 site
C A C N N N N G T G G T G N N N N C A C
PspXI  (39)
1 site
V C T C G A G B B G A G C T C V
SphI  (165)
1 site
G C A T G C C G T A C G
BsiWI  (702)
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.
RsrII  (762)
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.
BstEII  (780)
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.
SexAI  (1213)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be comp