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Plasmid Files

pWJ68

Bacterial plasmid for expressing catalytically inactive dCas9, with an N-terminal fusion to the RNA polymerase ω subunit for transcriptional activation.

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pWJ68 Sequence and MappWJ68.dna
Map and Sequence File   
Sequence Author:  Marraffini Lab / Addgene #46571
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 BaeGI - Bme1580I (9160) PflFI - Tth111I (9079) Bts α I (8996) Bsu36I (8970) AhdI (8586) BmeT110I (8298) AvaI - BsoBI (8297) PspFI (7825) BseYI (7821) EagI (7811) SalI (7523) EcoNI (7498) BciVI (6988) DR DR SacI (6480) Eco53kI (6478) PciI (4982) PfoI * (788) SacII (835) BssS α I (960) XbaI (1425) BspDI - ClaI (1519) BstEII (2284) SexAI * (2297) PstI (2523) BmgBI (2740) SwaI (3041) AleI (3767) PmlI (4813) pWJ68 9622 bp
BaeGI  (9160)
1 site
G K G C M C C M C G K G

Sticky ends from different BaeGI sites may not be compatible.
Bme1580I  (9160)
1 site
G K G C M C C M C G K G

Sticky ends from different Bme1580I sites may not be compatible.
PflFI  (9079)
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  (9079)
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.
BtsαI  (8996)
1 site
G C A G T G N N C G T C A C

Sticky ends from different BtsI sites may not be compatible.
Bsu36I  (8970)
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.
AhdI  (8586)
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.
BmeT110I  (8298)
1 site
C Y C G R G G R G C Y C
AvaI  (8297)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (8297)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures
up to 65°C.
PspFI  (7825)
1 site
C C C A G C G G G T C G
BseYI  (7821)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its
electrophoretic mobility.
EagI  (7811)
1 site
C G G C C G G C C G G C
SalI  (7523)
1 site
G T C G A C C A G C T G
EcoNI  (7498)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
BciVI  (6988)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
SacI  (6480)
1 site
G A G C T C C T C G A G
Eco53kI  (6478)
1 site
G A G C T C C T C G A G
PciI  (4982)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
PfoI  (788)
1 site
T C C N G G A A G G N C C T
* Blocked by Dcm methylation.
Sticky ends from different PfoI sites may not be compatible.
SacII  (835)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
BssSαI  (960)
1 site
C A C G A G G T G C T C
XbaI  (1425)
1 site
T C T A G A A G A T C T
BspDI  (1519)
1 site
A T C G A T T A G C T A
ClaI  (1519)
1 site
A T C G A T T A G C T A
BstEII  (2284)
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  (2297)
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.
PstI  (2523)
1 site
C T G C A G G A C G T C
BmgBI  (2740)
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.
SwaI  (3041)
1 site
A T T T A A A T T A A A T T T A

SwaI is typically used at 25°C, but is 50% active at 37°C.
AleI  (3767)
1 site
C A C N N N N G T G G T G N N N N C A C
PmlI  (4813)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
dCas9
2524 .. 6627  =  4104 bp
1367 amino acids  =  158.2 kDa
   Segment 1:  
   2524 .. 2547  =  24 bp
   8 amino acids  =  923.1 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
2524 .. 6627  =  4104 bp
1367 amino acids  =  158.2 kDa
   Segment 2:  
   2548 .. 2550  =  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
2524 .. 6627  =  4104 bp
1367 amino acids  =  158.2 kDa
   Segment 3:  
   2551 .. 5037  =  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
2524 .. 6627  =  4104 bp
1367 amino acids  =  158.2 kDa
   Segment 4:  
   5038 .. 5040  =  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
2524 .. 6627  =  4104 bp
1367 amino acids  =  158.2 kDa
   Segment 5:  
   5041 .. 6627  =  1587 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
2524 .. 6627  =  4104 bp
1367 amino acids  =  158.2 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
CmR
9182 .. 219  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
9182 .. 219  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
p15A ori
848 .. 1393  =  546 bp
Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
p15A ori
848 .. 1393  =  546 bp
Plasmids containing the medium-copy-number p15A
origin of replication can be propagated in E. coli cells
that contain a second plasmid with the ColE1 origin.
RNAP ω
2245 .. 2517  =  273 bp
91 amino acids  =  10.2 kDa
Product: DNA-directed RNA polymerase subunit
omega
promotes RNA polymerase assembly
RNAP ω
2245 .. 2517  =  273 bp
91 amino acids  =  10.2 kDa
Product: DNA-directed RNA polymerase subunit
omega
promotes RNA polymerase assembly
crRNA leader
6648 .. 6779  =  132 bp
crRNA leader sequence for the Streptococcus
pyogenes
CRISPR/Cas system
crRNA leader
6648 .. 6779  =  132 bp
crRNA leader sequence for the Streptococcus
pyogenes
CRISPR/Cas system
cat promoter
220 .. 322  =  103 bp
promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase
cat promoter
220 .. 322  =  103 bp
promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase
tracrRNA
1851 .. 1929  =  79 bp
trans-activating CRISPR RNA for the Streptococcus
pyogenes
CRISPR/Cas9 system
tracrRNA
1851 .. 1929  =  79 bp
trans-activating CRISPR RNA for the Streptococcus
pyogenes
CRISPR/Cas9 system
DR
6780 .. 6815  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR/Cas system
DR
6780 .. 6815  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR/Cas system
DR
6846 .. 6881  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR/Cas system
DR
6846 .. 6881  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR/Cas system
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