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Plasmid Files

pMJ806

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

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pMJ806 Sequence and MappMJ806.dna
Map and Sequence File   
Sequence Author:  Doudna Lab / Addgene #39312
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 BspQI - SapI (10,569) BssS α I (10,279) BspDI - ClaI (9561) SmaI (9380) TspMI - XmaI (9378) AsiSI (9256) DraIII (8557) PaeR7I - PspXI - XhoI (8150) EagI - NotI (8142) SalI (8129) PmlI (6277) KpnI (5762) Acc65I (5758) ScaI (5504) BstBI (5322) PflFI - Tth111I (130) PpuMI (867) FspAI (895) BglI (916) HpaI (1471) PspOMI (1766) ApaI (1770) NmeAIII (1773) BstEII (1791) SgrAI (2654) T7 promoter XbaI (2761) RBS ATG 6xHis BsaI (2887) BsiWI (3114) SpeI (3949) SwaI (4505) NheI (5083) BmtI (5087) AleI (5231) pMJ806 10,577 bp
BspQI  (10,569)
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  (10,569)
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  (10,279)
1 site
C A C G A G G T G C T C
BspDI  (9561)
1 site
A T C G A T T A G C T A
ClaI  (9561)
1 site
A T C G A T T A G C T A
SmaI  (9380)
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  (9378)
1 site
C C C G G G G G G C C C
XmaI  (9378)
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  (9256)
1 site
G C G A T C G C C G C T A G C G
DraIII  (8557)
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  (8150)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (8150)
1 site
V C T C G A G B B G A G C T C V
XhoI  (8150)
1 site
C T C G A G G A G C T C
EagI  (8142)
1 site
C G G C C G G C C G G C
NotI  (8142)
1 site
G C G G C C G C C G C C G G C G
SalI  (8129)
1 site
G T C G A C C A G C T G
PmlI  (6277)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
KpnI  (5762)
1 site
G G T A C C C C A T G G
Acc65I  (5758)
1 site
G G T A C C C C A T G G
ScaI  (5504)
1 site
A G T A C T T C A T G A
BstBI  (5322)
1 site
T T C G A A A A G C T T
PflFI  (130)
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  (130)
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.
PpuMI  (867)
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.
FspAI  (895)
1 site
R T G C G C A Y Y A C G C G T R
BglI  (916)
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.
HpaI  (1471)
1 site
G T T A A C C A A T T G
PspOMI  (1766)
1 site
G G G C C C C C C G G G
ApaI  (1770)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
NmeAIII  (1773)
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).
BstEII  (1791)
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.
SgrAI  (2654)
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.
XbaI  (2761)
1 site
T C T A G A A G A T C T
BsaI  (2887)
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  (3114)
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  (3949)
1 site
A C T A G T T G A T C A
SwaI  (4505)
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  (5083)
1 site
G C T A G C C G A T C G
BmtI  (5087)
1 site
G C T A G C C G A T C G
AleI  (5231)
1 site
C A C N N N N G T G G T G N N N N C A C
Cas9
3985 .. 8091  =  4107 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
3985 .. 8091  =  4107 bp
1368 amino acids  =  158.4 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes Type II CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
MBP
2827 .. 3924  =  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
2827 .. 3924  =  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
1246 .. 2328  =  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
1246 .. 2328  =  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
8871 .. 9686  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
8871 .. 9686  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
9808 .. 10,396  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
9808 .. 10,396  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
8324 .. 8779  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
8324 .. 8779  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
246 .. 437  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
246 .. 437  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
lacI promoter
2329 .. 2406  =  78 bp
lacI promoter
2329 .. 2406  =  78 bp
T7 terminator
8240 .. 8287  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
8240 .. 8287  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
2734 .. 2758  =  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
2734 .. 2758  =  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
2773 .. 2795  =  23 bp
efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)
RBS
2773 .. 2795  =  23 bp
efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)
TEV site
3955 .. 3975  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
TEV site
3955 .. 3975  =  21 bp
7 amino acids  =  870.0 Da
Product: tobacco etch virus (TEV) protease
recognition and cleavage site
T7 promoter
2715 .. 2733  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
2715 .. 2733  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
2806 .. 2823  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
2806 .. 2823  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
ATG
2803 .. 2805  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
2803 .. 2805  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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