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Plasmid Files

pET-Cas9-NLS-6xHis

Bacterial vector for regulated expression of S. pyogenes Cas9 with a C-terminal nuclear localization signal and 6xHis tag.

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pET-Cas9-NLS-6xHis Sequence and MappET-Cas9-NLS-6xHis.dna
Map and Sequence File   
Sequence Author:  Liu Lab / Addgene #62933
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 T7 promoter BspDI * - ClaI * (9228) SgrAI (9184) EcoNI (8971) ApaI (8300) PspOMI (8296) BssHII (8092) HpaI (8001) PpuMI (7634) PflFI - Tth111I (6897) BspQI - SapI (6756) AlwNI (6230) PvuI (5404) BglI (5154) BsaI (5095) AhdI (5034) XbaI (30) RBS BmgBI (290) SwaI (591) BfuAI - BspMI (1139) NheI (1169) BmtI (1173) AleI (1317) BstBI (1408) EcoRI (1410) BsmI (1674) Acc65I (1844) KpnI (1848) PmlI (2363) NruI (2401) BamHI (3448) Eco53kI (4028) SacI (4030) 6xHis PacI (4274) AvrII (4278) DraIII (4624) pET-Cas9-NLS-6xHis 9265 bp
BspDI  (9228)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
ClaI  (9228)
1 site
A T C G A T T A G C T A
* Blocked by Dam methylation.
SgrAI  (9184)
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.
EcoNI  (8971)
1 site
C C T N N N N N A G G G G A N N N N N T C C

The 1-base overhangs produced by EcoNI may be hard to ligate.
Sticky ends from different EcoNI sites may not be compatible.
ApaI  (8300)
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  (8296)
1 site
G G G C C C C C C G G G
BssHII  (8092)
1 site
G C G C G C C G C G C G

BssHII is typically used at 50°C, but is 75% active at 37°C.
HpaI  (8001)
1 site
G T T A A C C A A T T G
PpuMI  (7634)
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  (6897)
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  (6897)
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  (6756)
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  (6756)
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.
AlwNI  (6230)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
PvuI  (5404)
1 site
C G A T C G G C T A G C
BglI  (5154)
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.
BsaI  (5095)
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.
AhdI  (5034)
1 site
G A C N N N N N G T C C T G N N N N N C A G

The 1-base overhangs produced by AhdI may be hard to ligate.
Sticky ends from different AhdI sites may not be compatible.
XbaI  (30)
1 site
T C T A G A A G A T C T
BmgBI  (290)
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.
SwaI  (591)
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.
BfuAI  (1139)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI
recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (1139)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI
recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
NheI  (1169)
1 site
G C T A G C C G A T C G
BmtI  (1173)
1 site
G C T A G C C G A T C G
AleI  (1317)
1 site
C A C N N N N G T G G T G N N N N C A C
BstBI  (1408)
1 site
T T C G A A A A G C T T
EcoRI  (1410)
1 site
G A A T T C C T T A A G
BsmI  (1674)
1 site
G A A T G C N C T T A C G N

Sticky ends from different BsmI sites may not be compatible.
Acc65I  (1844)
1 site
G G T A C C C C A T G G
KpnI  (1848)
1 site
G G T A C C C C A T G G
PmlI  (2363)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
NruI  (2401)
1 site
T C G C G A A G C G C T
BamHI  (3448)
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.
Eco53kI  (4028)
1 site
G A G C T C C T C G A G
SacI  (4030)
1 site
G A G C T C C T C G A G
PacI  (4274)
1 site
T T A A T T A A A A T T A A T T
AvrII  (4278)
1 site
C C T A G G G G A T C C
DraIII  (4624)
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.
Cas9
71 .. 4174  =  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
71 .. 4174  =  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
7776 .. 8858  =  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
7776 .. 8858  =  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).
AmpR
4961 .. 5821  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   4961 .. 5752  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4961 .. 5821  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   5753 .. 5821  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
AmpR
4961 .. 5821  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and
related antibiotics
ori
5995 .. 6583  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
5995 .. 6583  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
f1 ori
4391 .. 4846  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
f1 ori
4391 .. 4846  =  456 bp
f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis
rop
7013 .. 7204  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
rop
7013 .. 7204  =  192 bp
63 amino acids  =  7.2 kDa
Product: Rop protein, which maintains plasmids at
low copy number
AmpR promoter
5822 .. 5914  =  93 bp
AmpR promoter
5822 .. 5914  =  93 bp
lacI promoter
8859 .. 8936  =  78 bp
lacI promoter
8859 .. 8936  =  78 bp
T7 terminator
4307 .. 4354  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
T7 terminator
4307 .. 4354  =  48 bp
transcription terminator for bacteriophage T7 RNA
polymerase
lac operator
3 .. 27  =  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
3 .. 27  =  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
42 .. 64  =  23 bp
efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)
RBS
42 .. 64  =  23 bp
efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)
SV40 NLS
4175 .. 4195  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4175 .. 4195  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
T7 promoter
9249 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
9249 .. 2  =  19 bp
promoter for bacteriophage T7 RNA polymerase
6xHis
4205 .. 4222  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
6xHis
4205 .. 4222  =  18 bp
6 amino acids  =  840.9 Da
Product: 6xHis affinity tag
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