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Plasmid Files

pME-Cas9

Middle entry Gateway® vector encoding zebrafish codon-optimized S. pyogenes Cas9 with nuclear localization signals.

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pME-Cas9 Sequence and MappME-Cas9.dna
Map and Sequence File   
Sequence Author:  Zon Lab / Addgene #63154
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 PciI (6901) BciVI (6703) ApaLI (6587) BsmBI (5742) AsiSI - PvuI (5726) SspI (5651) SacI (4971) Eco53kI (4969) BstXI (4963) AleI (4961) EagI - NotI (4950) XbaI (4943) BglII (4905) AgeI - SgrAI (4898) ScaI (4767) AarI (4760) AfeI (4439) KflI (4169) MscI * (3948) SexAI * (3363) BspQI - SapI (111) rrnB T2 terminator AclI (364) HpaI (501) AhdI (549) AflII (554) AbsI - PaeR7I - PspXI - XhoI (738) SalI (744) NcoI (754) BglI (756) ATG SbfI (1042) XcmI (1146) FspI (1209) NdeI (1604) Bsu36I (2061) DraI (2453) DraIII (3191) pME-Cas9 6907 bp
PciI  (6901)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BciVI  (6703)
1 site
G T A T C C ( N ) 5 N C A T A G G ( N ) 5

The 1-base overhangs produced by BciVI may be hard to ligate.
Sticky ends from different BciVI sites may not be compatible.
ApaLI  (6587)
1 site
G T G C A C C A C G T G
BsmBI  (5742)
1 site
C G T C T C N G C A G A G N ( N ) 4

Sticky ends from different BsmBI sites may not be compatible.
AsiSI  (5726)
1 site
G C G A T C G C C G C T A G C G
PvuI  (5726)
1 site
C G A T C G G C T A G C
SspI  (5651)
1 site
A A T A T T T T A T A A
SacI  (4971)
1 site
G A G C T C C T C G A G
Eco53kI  (4969)
1 site
G A G C T C C T C G A G
BstXI  (4963)
1 site
C C A N N N N N N T G G G G T N N N N N N A C C

Sticky ends from different BstXI sites may not be compatible.
AleI  (4961)
1 site
C A C N N N N G T G G T G N N N N C A C
EagI  (4950)
1 site
C G G C C G G C C G G C
NotI  (4950)
1 site
G C G G C C G C C G C C G G C G
XbaI  (4943)
1 site
T C T A G A A G A T C T
BglII  (4905)
1 site
A G A T C T T C T A G A
AgeI  (4898)
1 site
A C C G G T T G G C C A

AgeI quickly loses activity at 37°C, but can be used at 25°C for
long incubations.
SgrAI  (4898)
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.
ScaI  (4767)
1 site
A G T A C T T C A T G A
AarI  (4760)
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.
AfeI  (4439)
1 site
A G C G C T T C G C G A
KflI  (4169)
1 site
G G G W C C C C C C W G G G

Sticky ends from different KflI sites may not be compatible.
MscI  (3948)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
SexAI  (3363)
1 site
A C C W G G T T G G W C C A
* Blocked by Dcm methylation.
Sticky ends from different SexAI sites may not be compatible.
BspQI  (111)
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  (111)
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.
AclI  (364)
1 site
A A C G T T T T G C A A
HpaI  (501)
1 site
G T T A A C C A A T T G
AhdI  (549)
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.
AflII  (554)
1 site
C T T A A G G A A T T C

The sticky ends produced by AflII are hard to ligate.
AbsI  (738)
1 site
C C T C G A G G G G A G C T C C
PaeR7I  (738)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (738)
1 site
V C T C G A G B B G A G C T C V
XhoI  (738)
1 site
C T C G A G G A G C T C
SalI  (744)
1 site
G T C G A C C A G C T G
NcoI  (754)
1 site
C C A T G G G G T A C C
BglI  (756)
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.
SbfI  (1042)
1 site
C C T G C A G G G G A C G T C C
XcmI  (1146)
1 site
C C A N N N N N N N N N T G G G G T N N N N N N N N N A C C

The 1-base overhangs produced by XcmI may be hard to ligate.
Sticky ends from different XcmI sites may not be compatible.
FspI  (1209)
1 site
T G C G C A A C G C G T
NdeI  (1604)
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.
Bsu36I  (2061)
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.
DraI  (2453)
1 site
T T T A A A A A A T T T
DraIII  (3191)
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
795 .. 4895  =  4101 bp
1367 amino acids  =  158.2 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes MGAS15252 Type II
CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
Cas9
795 .. 4895  =  4101 bp
1367 amino acids  =  158.2 kDa
Product: Cas9 (Csn1) endonuclease from the
Streptococcus pyogenes MGAS15252 Type II
CRISPR/Cas system
generates RNA-guided double strand breaks in DNA
KanR
5301 .. 6110  =  810 bp
269 amino acids  =  30.8 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
5301 .. 6110  =  810 bp
269 amino acids  =  30.8 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
ori
6257 .. 6845  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
6257 .. 6845  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
attL1
569 .. 668  =  100 bp
recombination site for the Gateway® LR reaction
attL1
569 .. 668  =  100 bp
recombination site for the Gateway® LR reaction
attL2
5032 .. 5131  =  100 bp
recombination site for the Gateway® LR reaction
attL2
5032 .. 5131  =  100 bp
recombination site for the Gateway® LR reaction
rrnB T1 terminator
387 .. 473  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T1 terminator
387 .. 473  =  87 bp
transcription terminator T1 from the E. coli rrnB
gene
rrnB T2 terminator
268 .. 295  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
rrnB T2 terminator
268 .. 295  =  28 bp
transcription terminator T2 from the E. coli rrnB
gene
SV40 NLS
768 .. 788  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
768 .. 788  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4911 .. 4931  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4911 .. 4931  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
ATG
756 .. 758  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
756 .. 758  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
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