Resources
Plasmid Files

pHAGE EF1α dCas9-VP64

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

To see this sequence with restriction sites, features, and translations, please download
 SnapGene or the free  SnapGene Viewer.

pHAGE EF1-alpha dCas9-VP64 Sequence and MappHAGE EF1-alpha dCas9-VP64.dna
Map and Sequence File   
Sequence Author:  Maehr & Wolfe Labs / Addgene #50918
Download Free Trial Get SnapGene Viewer

 attB2 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 compatible.
TspMI  (2230)
1 site
C C C G G G G G G C C C
XmaI  (2230)
1 site
C C C G G G G G G C C C

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
SmaI  (2232)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
AhdI  (3255)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
SfiI  (4375)
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.
AvrII  (4422)
1 site
C C T A G G G G A T C C
dCas9
8340 .. 12,443  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   8340 .. 8366  =  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
8340 .. 12,443  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   8367 .. 8369  =  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
8340 .. 12,443  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   8370 .. 10,856  =  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
8340 .. 12,443  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   10,857 .. 10,859  =  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
8340 .. 12,443  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   10,860 .. 12,443  =  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
8340 .. 12,443  =  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
EF-1α promoter
7093 .. 8271  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
EF-1α promoter
7093 .. 8271  =  1179 bp
strong constitutive promoter for human elongation
factor EF-1α
AmpR
2468 .. 3328  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 1:  signal sequence  
   2468 .. 2536  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2468 .. 3328  =  861 bp
286 amino acids  =  31.5 kDa
   Segment 2:  
   2537 .. 3328  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
2468 .. 3328  =  861 bp
286 amino acids  =  31.5 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
LTR
4873 .. 5506  =  634 bp
long terminal repeat (LTR) from HIV-1
LTR
4873 .. 5506  =  634 bp
long terminal repeat (LTR) from HIV-1
PuroR
646 .. 1245  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
PuroR
646 .. 1245  =  600 bp
199 amino acids  =  21.5 kDa
Product: puromycin N-acetyltransferase
confers resistance to puromycin
WPRE
1262 .. 1850  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
WPRE
1262 .. 1850  =  589 bp
woodchuck hepatitis virus posttranscriptional
regulatory element
ori
3499 .. 4087  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
3499 .. 4087  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
PGK promoter
126 .. 625  =  500 bp
mouse phosphoglycerate kinase 1 promoter
PGK promoter
126 .. 625  =  500 bp
mouse phosphoglycerate kinase 1 promoter
LTR (ΔU3)
1925 .. 2158  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
LTR (ΔU3)
1925 .. 2158  =  234 bp
self-inactivating long terminal repeat (LTR) from
HIV-1
RRE
6175 .. 6408  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
RRE
6175 .. 6408  =  234 bp
The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to
the cytoplasm.
VP64
12,588 .. 12,737  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al.,
1998)
VP64
12,588 .. 12,737  =  150 bp
50 amino acids  =  5.5 kDa
Product: tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al.,
1998)
HIV-1 Ψ
5553 .. 5678  =  126 bp
packaging signal of human immunodeficiency virus
type 1
HIV-1 Ψ
5553 .. 5678  =  126 bp
packaging signal of human immunodeficiency virus
type 1
cPPT/CTS
6930 .. 7047  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
cPPT/CTS
6930 .. 7047  =  118 bp
central polypurine tract and central termination
sequence of HIV-1
AmpR promoter
2363 .. 2467  =  105 bp
AmpR promoter
2363 .. 2467  =  105 bp
3xHA
12,477 .. 12,566  =  90 bp
30 amino acids  =  3.5 kDa
Product: three tandem HA epitope tags
3xHA
12,477 .. 12,566  =  90 bp
30 amino acids  =  3.5 kDa
Product: three tandem HA epitope tags
attB1
8306 .. 8330  =  25 bp
recombination site for the Gateway® BP reaction
attB1
8306 .. 8330  =  25 bp
recombination site for the Gateway® BP reaction
attB2
12,784 .. 14  =  25 bp
recombination site for the Gateway® BP reaction
attB2
12,784 .. 14  =  25 bp
recombination site for the Gateway® BP reaction
SV40 NLS
12,456 .. 12,476  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
12,456 .. 12,476  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
EF-1α intron A
7324 .. 8262  =  939 bp
intron upstream of the start codon of human EF-1α
EF-1α intron A
7324 .. 8262  =  939 bp
intron upstream of the start codon of human EF-1α
Try SnapGene and create your own beautiful maps

Individual Sequences & Maps

SnapGene offers the fastest and easiest way to plan, visualize, and document your molecular biology procedures.

Priced accessibly so that everyone in your lab can have a license.

Learn More...

SnapGene Viewer is a versatile tool for creating and sharing richly annotated sequence files. It opens many common file formats.

Free! Because there should be no barriers to seeing your data.

Learn More...

The map, notes, and annotations on this page and in the sequence/map file are copyrighted material. This material may be used without restriction by academic, nonprofit, and governmental entities, except that the source must be cited as "www.snapgene.com/resources". Commercial entities must contact GSL Biotech LLC for permission and terms of use.

Copyright © 2016 GSL Biotech LLC | Site Map | Privacy | Legal Disclaimers   Subscribe to Our Newsletter