Resources
Plasmid Files

Cas9m4-VP64

Plasmid for expression in mammalian cells of catalytically dead Cas9m4 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.

Cas9m4-VP64 Sequence and MapCas9m4-VP64.dna
Map and Sequence File   
Sequence Author:  Church Lab / Addgene #47319
Download Free Trial Get SnapGene Viewer

 AhdI (8758) SgrDI (7832) MfeI (7669) MluI (7602) SpeI (7581) CMV enhancer SnaBI (7244) ATG SbfI (6625) XcmI (6518) BlpI (5917) Bsu36I (5599) BstZ17I (258) RsrII (713) AvrII (1438) StuI (1443) SexAI * (1670) DraIII (1968) PmeI (2510) AgeI (2524) AscI (2909) EcoRI (3042) PaeR7I - XhoI (3321) AarI (3604) FspAI (3918) PmlI (4079) BstXI (4083) BmgBI (4358) BamHI (4859) Cas9m4-VP64 9771 bp
AhdI  (8758)
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.
SgrDI  (7832)
1 site
C G T C G A C G G C A G C T G C
MfeI  (7669)
1 site
C A A T T G G T T A A C
MluI  (7602)
1 site
A C G C G T T G C G C A
SpeI  (7581)
1 site
A C T A G T T G A T C A
SnaBI  (7244)
1 site
T A C G T A A T G C A T
SbfI  (6625)
1 site
C C T G C A G G G G A C G T C C
XcmI  (6518)
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.
BlpI  (5917)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
Bsu36I  (5599)
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.
BstZ17I  (258)
1 site
G T A T A C C A T A T G
RsrII  (713)
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.
AvrII  (1438)
1 site
C C T A G G G G A T C C
StuI  (1443)
1 site
A G G C C T T C C G G A
SexAI  (1670)
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.
DraIII  (1968)
1 site
C A C N N N G T G G T G N N N C A C

Sticky ends from different DraIII sites may not be compatible.
PmeI  (2510)
1 site
G T T T A A A C C A A A T T T G
AgeI  (2524)
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.
AscI  (2909)
1 site
G G C G C G C C C C G C G C G G
EcoRI  (3042)
1 site
G A A T T C C T T A A G
PaeR7I  (3321)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (3321)
1 site
C T C G A G G A G C T C
AarI  (3604)
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  (3918)
1 site
R T G C G C A Y Y A C G C G T R
PmlI  (4079)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BstXI  (4083)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
BmgBI  (4358)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends
generated by BmgBI will not always regenerate a BmgBI site.
BamHI  (4859)
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.
Cas9m4
2766 .. 6869  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 7:  
   2766 .. 4280  =  1515 bp
   505 amino acids  =  58.3 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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
2766 .. 6869  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 6:  
   4281 .. 4283  =  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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
2766 .. 6869  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 5:  
   4284 .. 4349  =  66 bp
   22 amino acids  =  2.5 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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
2766 .. 6869  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 4:  
   4350 .. 4355  =  6 bp
   2 amino acids  =  160.2 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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
2766 .. 6869  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 3:  
   4356 .. 6839  =  2484 bp
   828 amino acids  =  96.1 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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
2766 .. 6869  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 2:  
   6840 .. 6842  =  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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
2766 .. 6869  =  4104 bp
1368 amino acids  =  158.2 kDa
   Segment 1:  
   6843 .. 6869  =  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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
Cas9m4
2766 .. 6869  =  4104 bp
1368 amino acids  =  158.2 kDa
7 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 mutations D10A,
D839A, H840A, and N863A (Mali et al., 2013)
AmpR
7971 .. 8831  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   7971 .. 8039  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7971 .. 8831  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   8040 .. 8831  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
7971 .. 8831  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
NeoR/KanR
567 .. 1361  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
567 .. 1361  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
ori
9002 .. 9590  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9002 .. 9590  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
1771 .. 2199  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
1771 .. 2199  =  429 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
CMV enhancer
7221 .. 7600  =  380 bp
human cytomegalovirus immediate early enhancer
CMV enhancer
7221 .. 7600  =  380 bp
human cytomegalovirus immediate early enhancer
SV40 promoter
1428 .. 1757  =  330 bp
SV40 enhancer and early promoter
SV40 promoter
1428 .. 1757  =  330 bp
SV40 enhancer and early promoter
CMV promoter
7017 .. 7220  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
CMV promoter
7017 .. 7220  =  204 bp
human cytomegalovirus (CMV) immediate early
promoter
VP64
2559 .. 2708  =  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
2559 .. 2708  =  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)
SV40 poly(A) signal
270 .. 391  =  122 bp
SV40 polyadenylation signal
SV40 poly(A) signal
270 .. 391  =  122 bp
SV40 polyadenylation signal
AmpR promoter
7866 .. 7970  =  105 bp
AmpR promoter
7866 .. 7970  =  105 bp
HSV TK poly(A) signal
2401 .. 2449  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
HSV TK poly(A) signal
2401 .. 2449  =  49 bp
herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)
SV40 NLS
2733 .. 2753  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
2733 .. 2753  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6879 .. 6899  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6879 .. 6899  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
6900 .. 6902  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
6900 .. 6902  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
SV40 ori
1442 .. 1577  =  136 bp
SV40 origin of replication
SV40 ori
1442 .. 1577  =  136 bp
SV40 origin of replication
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