Resources
Plasmid Files

pME-Cas9-T2A-GFP

Middle entry Gateway® vector encoding zebrafish codon-optimized S. pyogenes Cas9 with nuclear localization signals and with a C-terminal GFP.

To see this sequence with restriction sites, features, and translations, please download
 SnapGene or the free  SnapGene Viewer.

pME-Cas9-T2A-GFP Sequence and MappME-Cas9-T2A-GFP.dna
Map and Sequence File   
Sequence Author:  Zon Lab / Addgene #63155
Download Free Trial Get SnapGene Viewer

 PciI (7679) BciVI (7481) ApaLI (7365) BsmBI (6520) AsiSI - PvuI (6504) SspI (6429) SacI (5749) Eco53kI (5747) BstXI (5741) SacII (5740) EagI - NotI (5728) BtgZI (5128) XbaI (5001) BamHI (4905) AgeI - SgrAI (4898) ScaI (4767) AarI (4760) AfeI (4439) KflI (4169) 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) SexAI * (3363) MscI * (3948) pME-Cas9-T2A-GFP 7685 bp
PciI  (7679)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
BciVI  (7481)
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  (7365)
1 site
G T G C A C C A C G T G
BsmBI  (6520)
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  (6504)
1 site
G C G A T C G C C G C T A G C G
PvuI  (6504)
1 site
C G A T C G G C T A G C
SspI  (6429)
1 site
A A T A T T T T A T A A
SacI  (5749)
1 site
G A G C T C C T C G A G
Eco53kI  (5747)
1 site
G A G C T C C T C G A G
BstXI  (5741)
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.
SacII  (5740)
1 site
C C G C G G G G C G C C

Efficient cleavage requires at least two copies of the SacII
recognition sequence.
EagI  (5728)
1 site
C G G C C G G C C G G C
NotI  (5728)
1 site
G C G G C C G C C G C C G G C G
BtgZI  (5128)
1 site
G C G A T G ( N ) 10 C G C T A C ( N ) 10 ( N ) 4

Sticky ends from different BtgZI sites may not be compatible.
After cleavage, BtgZI can remain bound to DNA and alter its
electrophoretic mobility.
BtgZI is typically used at 60°C, but is 75% active at 37°C.
XbaI  (5001)
1 site
T C T A G A A G A T C T
BamHI  (4905)
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.
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.
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.
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.
MscI  (3948)
1 site
T G G C C A A C C G G T
* Blocked by Dcm methylation.
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
6079 .. 6888  =  810 bp
269 amino acids  =  30.8 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
KanR
6079 .. 6888  =  810 bp
269 amino acids  =  30.8 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin in bacteria or G418
(Geneticin®) in eukaryotes
EGFP
5007 .. 5726  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 1:  
   5007 .. 5009  =  3 bp
   1 amino acid  =  149.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
5007 .. 5726  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 2:  1a  
   5010 .. 5012  =  3 bp
   1 amino acid  =  117.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
5007 .. 5726  =  720 bp
239 amino acids  =  26.9 kDa
   Segment 3:  
   5013 .. 5726  =  714 bp
   237 amino acids  =  26.7 kDa
Product: enhanced GFP
mammalian codon-optimized
EGFP
5007 .. 5726  =  720 bp
239 amino acids  =  26.9 kDa
3 segments
Product: enhanced GFP
mammalian codon-optimized
ori
7035 .. 7623  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7035 .. 7623  =  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
5810 .. 5909  =  100 bp
recombination site for the Gateway® LR reaction
attL2
5810 .. 5909  =  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
T2A
4947 .. 5000  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
T2A
4947 .. 5000  =  54 bp
18 amino acids  =  1.8 kDa
Product: 2A peptide from Thosea asigna virus capsid
protein
Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides.
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
Try SnapGene and create your own beautiful maps

Individual Sequences & Maps

SnapGene offers the fastest and easiest way to plan, visualize, and document your molecular biology procedures.

Priced accessibly so that everyone in your lab can have a license.

Learn More...

SnapGene Viewer is a versatile tool for creating and sharing richly annotated sequence files. It opens many common file formats.

Free! Because there should be no barriers to seeing your data.

Learn More...

The map, notes, and annotations on this page and in the sequence/map file are copyrighted material. This material may be used without restriction by academic, nonprofit, and governmental entities, except that the source must be cited as "www.snapgene.com/resources". Commercial entities must contact GSL Biotech LLC for permission and terms of use.

Copyright © 2016 GSL Biotech LLC | Site Map | Privacy | Legal Disclaimers   Subscribe to Our Newsletter