pMJ841

pET-derived bacterial vector for expressing 6xHis- and MBP-tagged catalytically inactive S. pyogenes Cas9.

Sequence Author: Doudna Lab / Addgene #39318

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T7 promoter RBS ATG 6xHis pMJ841 10,577 bp
ATG
118 .. 120  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
118 .. 120  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
6xHis
121 .. 138  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
121 .. 138  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
MBP
142 .. 1239  =  1098 bp
366 amino acids  =  40.2 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
MBP
142 .. 1239  =  1098 bp
366 amino acids  =  40.2 kDa
Product: maltose binding protein from E. coli
This version of the gene does not encode a signal sequence, so MBP will remain in the cytosol.
TEV site
1270 .. 1290  =  21 bp
7 amino acids  =  869.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
1270 .. 1290  =  21 bp
7 amino acids  =  869.9 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
dCas9
1300 .. 5406  =  4107 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 1:  
   1300 .. 1326  =  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
1300 .. 5406  =  4107 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 2:  
   1327 .. 1329  =  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
1300 .. 5406  =  4107 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 3:  
   1330 .. 3816  =  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
1300 .. 5406  =  4107 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 4:  
   3817 .. 3819  =  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
1300 .. 5406  =  4107 bp
1368 amino acids  =  158.3 kDa
5 segments
   Segment 5:  
   3820 .. 5406  =  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
1300 .. 5406  =  4107 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
lacI
9138 .. 10,220  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lacI
9138 .. 10,220  =  1083 bp
360 amino acids  =  38.6 kDa
Product: lac repressor
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
KanR
6186 .. 7001  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
KanR
6186 .. 7001  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418 (Geneticin®) in eukaryotes
ori
7123 .. 7711  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
7123 .. 7711  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
f1 ori
5639 .. 6094  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
f1 ori
5639 .. 6094  =  456 bp
f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis
rop
8138 .. 8329  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
rop
8138 .. 8329  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at low copy number
lacI promoter
10,221 .. 10,298  =  78 bp
lacI promoter
10,221 .. 10,298  =  78 bp
T7 terminator
5555 .. 5602  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
T7 terminator
5555 .. 5602  =  48 bp
transcription terminator for bacteriophage T7 RNA polymerase
lac operator
49 .. 73  =  25 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
lac operator
49 .. 73  =  25 bp
The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-β-D-thiogalactopyranoside (IPTG).
RBS
88 .. 110  =  23 bp
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
RBS
88 .. 110  =  23 bp
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)
T7 promoter
30 .. 48  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
30 .. 48  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ORF:  118 .. 5406  =  5289 bp
ORF:  1762 amino acids  =  201.6 kDa
ORF:  10,396 .. 58  =  240 bp
ORF:  79 amino acids  =  8.0 kDa
ORF:  9104 .. 9355  =  252 bp
ORF:  83 amino acids  =  9.1 kDa
ORF:  10,259 .. 10,522  =  264 bp
ORF:  87 amino acids  =  9.5 kDa
ORF:  8361 .. 8729  =  369 bp
ORF:  122 amino acids  =  14.2 kDa
ORF:  6186 .. 7001  =  816 bp
ORF:  271 amino acids  =  31.0 kDa
ORF:  9138 .. 10,097  =  960 bp
ORF:  319 amino acids  =  34.1 kDa
ORF:  8138 .. 8362  =  225 bp
ORF:  74 amino acids  =  8.5 kDa
ORF:  8726 .. 9082  =  357 bp
ORF:  118 amino acids  =  13.0 kDa
ORF:  10,467 .. 258  =  369 bp
ORF:  122 amino acids  =  13.3 kDa
ORF:  9118 .. 9381  =  264 bp
ORF:  87 amino acids  =  8.9 kDa
ORF:  9970 .. 10,470  =  501 bp
ORF:  166 amino acids  =  17.5 kDa
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