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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.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Kamoun Lab / Addgene #46965
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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) ZraI (4428) AatII (4430) PspOMI (4432) ApaI (4436) XbaI (4438) Acc65I (4453) KpnI (4457) RsrII (5409) MauBI (5823) 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
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
ORF:  4765 .. 5559  =  795 bp
ORF:  264 amino acids  =  29.0 kDa
ORF:  6355 .. 7287  =  933 bp
ORF:  310 amino acids  =  34.5 kDa
ORF:  9682 .. 10,275  =  594 bp
ORF:  197 amino acids  =  22.2 kDa
ORF:  11,782 .. 12,132  =  351 bp
ORF:  116 amino acids  =  12.6 kDa
ORF:  12,703 .. 12,966  =  264 bp
ORF:  87 amino acids  =  10.4 kDa
ORF:  13,468 .. 4140  =  5073 bp
ORF:  1690 amino acids  =  194.9 kDa
ORF:  4937 .. 5323  =  387 bp
ORF:  128 amino acids  =  14.6 kDa
ORF:  10,019 .. 10,282  =  264 bp
ORF:  87 amino acids  =  9.1 kDa
ORF:  11,441 .. 11,713  =  273 bp
ORF:  90 amino acids  =  9.9 kDa
ORF:  11,127 .. 11,441  =  315 bp
ORF:  104 amino acids  =  12.0 kDa
ORF:  12,624 .. 13,442  =  819 bp
ORF:  272 amino acids  =  30.9 kDa
ORF:  13,507 .. 480  =  1374 bp
ORF:  457 amino acids  =  47.9 kDa
ORF:  1405 .. 1923  =  519 bp
ORF:  172 amino acids  =  20.1 kDa
ORF:  1987 .. 2520  =  534 bp
ORF:  177 amino acids  =  19.9 kDa
ORF:  3514 .. 4047  =  534 bp
ORF:  177 amino acids  =  20.0 kDa
ORF:  5890 .. 6231  =  342 bp
ORF:  113 amino acids  =  12.1 kDa
ORF:  6535 .. 6918  =  384 bp
ORF:  127 amino acids  =  14.6 kDa
ORF:  9049 .. 10,155  =  1107 bp
ORF:  368 amino acids  =  41.2 kDa
ORF:  14,283 .. 143  =  261 bp
ORF:  86 amino acids  =  9.7 kDa
ORF:  6528 .. 6785  =  258 bp
ORF:  85 amino acids  =  9.1 kDa
ORF:  6795 .. 7073  =  279 bp
ORF:  92 amino acids  =  9.4 kDa
ORF:  11,478 .. 12,164  =  687 bp
ORF:  228 amino acids  =  25.1 kDa
ORF:  12,651 .. 12,950  =  300 bp
ORF:  99 amino acids  =  11.2 kDa
ORF:  6101 .. 6343  =  243 bp
ORF:  80 amino acids  =  9.1 kDa
ORF:  10,550 .. 11,179  =  630 bp
ORF:  209 amino acids  =  22.1 kDa
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