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Plasmid Files

pWJ66

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

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pWJ66 Sequence and MappWJ66.dna
Map and Sequence File   
Sequence Author:  Marraffini Lab / Addgene #46570
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 BsaI (6873) DR BsaI (6776) BsaI (6741) DR pWJ66 9552 bp
BsaI  (6873)
3 sites
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
BsaI  (6776)
3 sites
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
BsaI  (6741)
3 sites
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
dCas9
2225 .. 6328  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 1:  
   2225 .. 2251  =  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
2225 .. 6328  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 2:  
   2252 .. 2254  =  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
2225 .. 6328  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 3:  
   2255 .. 4741  =  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
2225 .. 6328  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 4:  
   4742 .. 4744  =  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
2225 .. 6328  =  4104 bp
1368 amino acids  =  158.3 kDa
   Segment 5:  
   4745 .. 6328  =  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
2225 .. 6328  =  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
CmR
9112 .. 219  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
9112 .. 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
6335 .. 6607  =  273 bp
90 amino acids  =  10.1 kDa
Product: DNA-directed RNA polymerase subunit
omega
promotes RNA polymerase assembly
RNAP ω
6335 .. 6607  =  273 bp
90 amino acids  =  10.1 kDa
Product: DNA-directed RNA polymerase subunit
omega
promotes RNA polymerase assembly
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
crRNA leader
6608 .. 6709  =  102 bp
crRNA leader sequence for the Streptococcus
pyogenes
CRISPR/Cas system
crRNA leader
6608 .. 6709  =  102 bp
crRNA leader sequence for the Streptococcus
pyogenes
CRISPR/Cas system
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
6710 .. 6745  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR/Cas system
DR
6710 .. 6745  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR/Cas system
DR
6776 .. 6811  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR/Cas system
DR
6776 .. 6811  =  36 bp
direct repeat for the Streptococcus pyogenes
CRISPR/Cas system
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