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Plasmid Files

pET-28b-Cas9-His

Bacterial plasmid for expressing a 6xHis-tagged Cas9 protein, which can be purified and microinjected for gene editing.

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pET-28b-Cas9-His Sequence and MappET-28b-Cas9-His.dna
Map and Sequence File   
Sequence Author:  Schier Lab / Addgene #47327
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 BspQI - SapI (9367) BspDI - ClaI (8359) AsiSI (8054) DraIII (7355) 6xHis PspXI (6948) NotI (6940) AscI (6757) EcoRI (6624) AarI (6062) PmlI (5591) BmgBI (5312) BamHI (4807) BstZ17I (105) BglI (916) PshAI (1132) HpaI (1471) PspOMI (1766) ApaI (1770) BstEII (1791) MluI (1973) BstAPI (2297) SphI (2506) SgrAI (2654) T7 promoter XbaI (2761) RBS NcoI (2800) SbfI (3049) NdeI (3280) SalI (3342) DraI (3557) Bsu36I (4068) Eco53kI (4370) SacI (4372) pET-28b-Cas9-His 9375 bp
BspQI  (9367)
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  (9367)
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.
BspDI  (8359)
1 site
A T C G A T T A G C T A
ClaI  (8359)
1 site
A T C G A T T A G C T A
AsiSI  (8054)
1 site
G C G A T C G C C G C T A G C G
DraIII  (7355)
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.
PspXI  (6948)
1 site
V C T C G A G B B G A G C T C V
NotI  (6940)
1 site
G C G G C C G C C G C C G G C G
AscI  (6757)
1 site
G G C G C G C C C C G C G C G G
EcoRI  (6624)
1 site
G A A T T C C T T A A G
AarI  (6062)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI
recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its
electrophoretic mobility.
PmlI  (5591)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
BmgBI  (5312)
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.
BamHI  (4807)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can
remain bound to DNA and alter its electrophoretic mobility.
BstZ17I  (105)
1 site
G T A T A C C A T A T G
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.
PshAI  (1132)
1 site
G A C N N N N G T C C T G N N N N C A G

PshAI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
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.
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.
MluI  (1973)
1 site
A C G C G T T G C G C A
BstAPI  (2297)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
SphI  (2506)
1 site
G C A T G C C G T A C G
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
NcoI  (2800)
1 site
C C A T G G G G T A C C
SbfI  (3049)
1 site
C C T G C A G G G G A C G T C C
NdeI  (3280)
1 site
C A T A T G G T A T A C

Prolonged incubation with NdeI may lead to removal of additional
nucleotides.
SalI  (3342)
1 site
G T C G A C C A G C T G
DraI  (3557)
1 site
T T T A A A A A A T T T
Bsu36I  (4068)
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.
Eco53kI  (4370)
1 site
G A G C T C C T C G A G
SacI  (4372)
1 site
G A G C T C C T C G A G
Cas9
2802 .. 6905  =  4104 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
2802 .. 6905  =  4104 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
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
7669 .. 8484  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
7669 .. 8484  =  816 bp
271 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
8606 .. 9194  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
8606 .. 9194  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
7122 .. 7577  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
7122 .. 7577  =  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
bom
5 .. 144  =  140 bp
basis of mobility region from pBR322
bom
5 .. 144  =  140 bp
basis of mobility region from pBR322
lacI promoter
2329 .. 2406  =  78 bp
lacI promoter
2329 .. 2406  =  78 bp
T7 terminator
7038 .. 7085  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
7038 .. 7085  =  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)
SV40 NLS
6918 .. 6938  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
6918 .. 6938  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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
6954 .. 6971  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
6954 .. 6971  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
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