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Plasmid Files

pK7WGF2::hCas9

Plant vector for expressing human codon-optimized Cas9 with an N-terminal enhanced GFP (EGFP) tag.

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pK7WGF2_hCas9 Sequence and MappK7WGF2_hCas9.dna
Map and Sequence File   
Sequence Author:  Kamoun Lab / Addgene #46965
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 attB1 SpeI (13,627) PshAI (13,357) StuI (12,762) PmeI (12,517) BsiWI (9502) AgeI (8892) BstZ17I (8406) HpaI (7333) BstXI - PmlI (2790) FspAI (2951) NruI (3241) AarI (3261) PaeR7I - XhoI (3544) EcoRI (3823) SV40 NLS ZraI (4428) AatII (4430) PspOMI (4432) ApaI (4436) XbaI (4438) Acc65I (4453) KpnI (4457) RsrII (5409) MauBI (5823) LB T-DNA repeat BstEII (6654) pK7WGF2::hCas9 14,400 bp
SpeI  (13,627)
1 site
A C T A G T T G A T C A
PshAI  (13,357)
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.
StuI  (12,762)
1 site
A G G C C T T C C G G A
PmeI  (12,517)
1 site
G T T T A A A C C A A A T T T G
BsiWI  (9502)
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.
AgeI  (8892)
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.
BstZ17I  (8406)
1 site
G T A T A C C A T A T G
HpaI  (7333)
1 site
G T T A A C C A A T T G
BstXI  (2790)
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.
PmlI  (2790)
1 site
C A C G T G G T G C A C

PmlI gradually loses activity when stored at -20°C.
FspAI  (2951)
1 site
R T G C G C A Y Y A C G C G T R
NruI  (3241)
1 site
T C G C G A A G C G C T
AarI  (3261)
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.
PaeR7I  (3544)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (3544)
1 site
C T C G A G G A G C T C
EcoRI  (3823)
1 site
G A A T T C C T T A A G
ZraI  (4428)
1 site
G A C G T C C T G C A G
AatII  (4430)
1 site
G A C G T C C T G C A G
PspOMI  (4432)
1 site
G G G C C C C C C G G G
ApaI  (4436)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
XbaI  (4438)
1 site
T C T A G A A G A T C T
Acc65I  (4453)
1 site
G G T A C C C C A T G G
KpnI  (4457)
1 site
G G T A C C C C A T G G
RsrII  (5409)
1 site
C G G W C C G G C C W G G C

Efficient cleavage requires at least two copies of the RsrII
recognition sequence.
Sticky ends from different RsrII sites may not be compatible.
For full activity, add fresh DTT.
MauBI  (5823)
1 site
C G C G C G C G G C G C G C G C
BstEII  (6654)
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.
Cas9
1 .. 4104  =  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
1 .. 4104  =  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
pVS1 RepA
9049 .. 10,116  =  1068 bp
355 amino acids  =  39.7 kDa
Product: replication protein from the Pseudomonas
plasmid pVS1 (Heeb et al., 2000)
pVS1 RepA
9049 .. 10,116  =  1068 bp
355 amino acids  =  39.7 kDa
Product: replication protein from the Pseudomonas
plasmid pVS1 (Heeb et al., 2000)
NeoR/KanR
4765 .. 5559  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
NeoR/KanR
4765 .. 5559  =  795 bp
264 amino acids  =  29.0 kDa
Product: aminoglycoside phosphotransferase from
Tn5
confers resistance to neomycin, kanamycin, and
G418 (Geneticin®)
SmR
6496 .. 7287  =  792 bp
263 amino acids  =  29.2 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
SmR
6496 .. 7287  =  792 bp
263 amino acids  =  29.2 kDa
Product: aminoglycoside adenylyltransferase
(Murphy, 1985)
confers resistance to spectinomycin and
streptomycin
EGFP
13,633 .. 14,349  =  717 bp
239 amino acids  =  26.9 kDa
   Segment 1:  
   13,633 .. 13,635  =  3 bp
   1 amino acid  =  149.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
13,633 .. 14,349  =  717 bp
239 amino acids  =  26.9 kDa
   Segment 2:  1a  
   13,636 .. 13,638  =  3 bp
   1 amino acid  =  117.2 Da
Product: enhanced GFP
mammalian codon-optimized
EGFP
13,633 .. 14,349  =  717 bp
239 amino acids  =  26.9 kDa
   Segment 3:  
   13,639 .. 14,349  =  711 bp
   237 amino acids  =  26.7 kDa
Product: enhanced GFP
mammalian codon-optimized
EGFP
13,633 .. 14,349  =  717 bp
239 amino acids  =  26.9 kDa
3 segments
Product: enhanced GFP
mammalian codon-optimized
pVS1 StaA
10,550 .. 11,179  =  630 bp
209 amino acids  =  22.1 kDa
Product: stability protein from the Pseudomonas
plasmid pVS1 (Heeb et al., 2000)
pVS1 StaA
10,550 .. 11,179  =  630 bp
209 amino acids  =  22.1 kDa
Product: stability protein from the Pseudomonas
plasmid pVS1 (Heeb et al., 2000)
ori
7531 .. 8119  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
ori
7531 .. 8119  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin
of replication
CaMV 35S promoter
13,196 .. 13,540  =  345 bp
strong constitutive promoter from cauliflower
mosaic virus
CaMV 35S promoter
13,196 .. 13,540  =  345 bp
strong constitutive promoter from cauliflower
mosaic virus
NOS terminator
5599 .. 5851  =  253 bp
nopaline synthase terminator and poly(A) signal
NOS terminator
5599 .. 5851  =  253 bp
nopaline synthase terminator and poly(A) signal
pVS1 oriV
8789 .. 8983  =  195 bp
origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)
pVS1 oriV
8789 .. 8983  =  195 bp
origin of replication for the Pseudomonas plasmid
pVS1 (Heeb et al., 2000)
NOS promoter
4552 .. 4731  =  180 bp
nopaline synthase promoter
NOS promoter
4552 .. 4731  =  180 bp
nopaline synthase promoter
bom
8305 .. 8445  =  141 bp
basis of mobility region from pBR322
bom
8305 .. 8445  =  141 bp
basis of mobility region from pBR322
attB2
4156 .. 4180  =  25 bp
recombination site for the Gateway® BP reaction
attB2
4156 .. 4180  =  25 bp
recombination site for the Gateway® BP reaction
LB T-DNA repeat
5944 .. 5968  =  25 bp
left border repeat from nopaline C58 T-DNA
LB T-DNA repeat
5944 .. 5968  =  25 bp
left border repeat from nopaline C58 T-DNA
RB T-DNA repeat
12,479 .. 12,503  =  25 bp
right border repeat from nopaline C58 T-DNA
RB T-DNA repeat
12,479 .. 12,503  =  25 bp
right border repeat from nopaline C58 T-DNA
attB1
14,356 .. 14,380  =  25 bp
recombination site for the Gateway® BP reaction
attB1
14,356 .. 14,380  =  25 bp
recombination site for the Gateway® BP reaction
SV40 NLS
4117 .. 4137  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
SV40 NLS
4117 .. 4137  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T
antigen
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