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pMJ841

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

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pMJ841.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Doudna Lab / Addgene #39318
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SgrAI (10,546) BstEII (9683) NmeAIII (9665) ApaI (9662) PspOMI (9658) HpaI (9363) BglI (8808) FspAI (8787) PpuMI (8759) PflFI - Tth111I (8022) BspQI - SapI (7884) BssS α I (7594) BspDI - ClaI (6876) SmaI (6695) TspMI - XmaI (6693) AsiSI (6571) DraIII (5872) PaeR7I - PspXI - XhoI (5465) EagI - NotI (5457) XbaI (76) BsaI (202) BsiWI (429) SpeI (1264) SwaI (1820) NheI (2398) BmtI (2402) AleI (2546) BstBI (2637) ScaI (2819) Acc65I (3073) KpnI (3077) PmlI (3592) SalI (5444) pMJ841 10,577 bp
SgrAI  (10,546)
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.
BstEII  (9683)
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.
NmeAIII  (9665)
1 site
G C C G A G ( N ) 18-19 N N C G G C T C ( N ) 18-19

Efficient cleavage requires at least two copies of the NmeAIII recognition sequence.
Sticky ends from different NmeAIII sites may not be compatible.
For full activity, add fresh S-adenosylmethionine (SAM).
ApaI  (9662)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
PspOMI  (9658)
1 site
G G G C C C C C C G G G
HpaI  (9363)
1 site
G T T A A C C A A T T G
BglI  (8808)
1 site
G C C N N N N N G G C C G G N N N N N C C G

Sticky ends from different BglI sites may not be compatible.
FspAI  (8787)
1 site
R T G C G C A Y Y A C G C G T R
PpuMI  (8759)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
PflFI  (8022)
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  (8022)
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.
BspQI  (7884)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different BspQI sites may not be compatible.
SapI  (7884)
1 site
G C T C T T C N C G A G A A G N N N N

Sticky ends from different SapI sites may not be compatible.
SapI gradually settles in solution, so a tube of SapI should be mixed before removing an aliquot.
BssSαI  (7594)
1 site
C A C G A G G T G C T C
BspDI  (6876)
1 site
A T C G A T T A G C T A
ClaI  (6876)
1 site
A T C G A T T A G C T A
SmaI  (6695)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
TspMI  (6693)
1 site
C C C G G G G G G C C C
XmaI  (6693)
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.
AsiSI  (6571)
1 site
G C G A T C G C C G C T A G C G
DraIII  (5872)
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.
PaeR7I  (5465)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (5465)
1 site
V C T C G A G B B G A G C T C V
XhoI  (5465)
1 site
C T C G A G G A G C T C
EagI  (5457)
1 site
C G G C C G G C C G G C
NotI  (5457)
1 site
G C G G C C G C C G C C G G C G
XbaI  (76)
1 site
T C T A G A A G A T C T
BsaI  (202)
1 site
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.
BsiWI  (429)
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.
SpeI  (1264)
1 site
A C T A G T T G A T C A
SwaI  (1820)
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.
NheI  (2398)
1 site
G C T A G C C G A T C G
BmtI  (2402)
1 site
G C T A G C C G A T C G
AleI  (2546)
1 site
C A C N N N N G T G G T G N N N N C A C
BstBI  (2637)
1 site
T T C G A A A A G C T T
ScaI  (2819)
1 site
A G T A C T T C A T G A
Acc65I  (3073)
1 site
G G T A C C C C A T G G
KpnI  (3077)
1 site
G G T A C C C C A T G G
PmlI  (3592)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
SalI  (5444)
1 site
G T C G A C C A G C T G
dCas9
1300 .. 5406  =  4107 bp
1368 amino acids  =  158.3 kDa
   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
   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
   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
   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
   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
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.
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)
TEV site
1270 .. 1290  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
TEV site
1270 .. 1290  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease recognition and cleavage site
T7 promoter
30 .. 48  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
30 .. 48  =  19 bp
promoter for bacteriophage T7 RNA polymerase
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
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
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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