pFGC-pcoCas9

Binary plant vector for expressing plant codon-optimized Case9 from the 35SPPDK promoter.

Sequence Author: Sheen Lab / Addgene #52256

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AanI (11,623) LB T-DNA repeat EcoRI (9934) PaeR7I - PspXI - XhoI (9928) SnaBI (9924) SwaI (9911) XbaI (9902) AscI (9895) I-PpoI (9882) PacI (9863) I-SceI (9853) I-CeuI (9826) Bsu36I (9822) PI-PspI (9801) SbfI (9751) NruI (9742) AsiSI (9736) SmaI - SrfI (9721) TspMI - XmaI (9719) NOS terminator AanI (6692) NcoI (5003) ATG 2xFLAG SV40 NLS AanI (6670) pFGC-pcoCas9 13,330 bp
AanI  (11,623)
3 sites
T T A T A A A A T A T T
EcoRI  (9934)
1 site
G A A T T C C T T A A G
PaeR7I  (9928)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
PspXI  (9928)
1 site
V C T C G A G B B G A G C T C V
XhoI  (9928)
1 site
C T C G A G G A G C T C
SnaBI  (9924)
1 site
T A C G T A A T G C A T
SwaI  (9911)
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.
XbaI  (9902)
1 site
T C T A G A A G A T C T
AscI  (9895)
1 site
G G C G C G C C C C G C G C G G
I-PpoI  (9882)
1 site
C T C T C T T A A G G T A G C G A G A G A A T T C C A T C G

I-PpoI is a homing endonuclease that can recognize a variety of similar recognition sequences.
PacI  (9863)
1 site
T T A A T T A A A A T T A A T T
I-SceI  (9853)
1 site
T A G G G A T A A C A G G G T A A T A T C C C T A T T G T C C C A T T A

I-SceI is a homing endonuclease that can recognize a variety of similar recognition sequences.
After cleavage, I-SceI can remain bound to DNA and alter its electrophoretic mobility.
I-CeuI  (9826)
1 site
T A A C T A T A A C G G T C C T A A G G T A G C G A A T T G A T A T T G C C A G G A T T C C A T C G C T

I-CeuI is a homing endonuclease that can recognize a variety of similar recognition sequences.
Bsu36I  (9822)
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.
PI-PspI  (9801)
1 site
T G G C A A A C A G C T A T T A T G G G T A T T A T G G G T A C C G T T T G T C G A T A A T A C C C A T A A T A C C C A

PI-PspI is a homing endonuclease that can recognize a variety of similar recognition sequences.
SbfI  (9751)
1 site
C C T G C A G G G G A C G T C C
NruI  (9742)
1 site
T C G C G A A G C G C T
AsiSI  (9736)
1 site
G C G A T C G C C G C T A G C G
SmaI  (9721)
1 site
C C C G G G G G G C C C

SmaI can be used at 37°C for brief incubations.
SrfI  (9721)
1 site
G C C C G G G C C G G G C C C G
TspMI  (9719)
1 site
C C C G G G G G G C C C
XmaI  (9719)
1 site
C C C G G G G G G C C C

Cleavage may be enhanced when more than one copy of the XmaI recognition sequence is present.
AanI  (6692)
3 sites
T T A T A A A A T A T T
NcoI  (5003)
1 site
C C A T G G G G T A C C
AanI  (6670)
3 sites
T T A T A A A A T A T T
ATG
5005 .. 5007  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
5005 .. 5007  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
2xFLAG
5008 .. 5055  =  48 bp
16 amino acids  =  2.0 kDa
Product: two tandem FLAG® epitope tags
2xFLAG
5008 .. 5055  =  48 bp
16 amino acids  =  2.0 kDa
Product: two tandem FLAG® epitope tags
SV40 NLS
5062 .. 5082  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
5062 .. 5082  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
pcoCas9
5107 .. 9396  =  4290 bp
1367 amino acids  =  158.3 kDa
2 segments  =  4101 bp
Product: Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
plant codon-optimized Cas9 gene containing the potato IV2 intron
pcoCas9
5107 .. 9396  =  4290 bp
1367 amino acids  =  158.3 kDa
2 segments  =  4101 bp
   Segment 1:  
   5107 .. 6543  =  1437 bp
   479 amino acids  =  55.4 kDa
Product: Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
plant codon-optimized Cas9 gene containing the potato IV2 intron
pcoCas9
5107 .. 9396  =  4290 bp
1367 amino acids  =  158.3 kDa
2 segments  =  4101 bp
   Segment 2:  
   6733 .. 9396  =  2664 bp
   888 amino acids  =  102.9 kDa
Product: Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
plant codon-optimized Cas9 gene containing the potato IV2 intron
pcoCas9
5107 .. 9396  =  4290 bp
1367 amino acids  =  158.3 kDa
2 segments  =  4101 bp
Product: Cas9 endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system
plant codon-optimized Cas9 gene containing the potato IV2 intron
nucleoplasmin NLS
9397 .. 9444  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from nucleoplasmin
nucleoplasmin NLS
9397 .. 9444  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from nucleoplasmin
pVS1 RepA
749 .. 1822  =  1074 bp
357 amino acids  =  39.9 kDa
Product: replication protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)
pVS1 RepA
749 .. 1822  =  1074 bp
357 amino acids  =  39.9 kDa
Product: replication protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)
KanR
11,680 .. 12,474  =  795 bp
264 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
KanR
11,680 .. 12,474  =  795 bp
264 amino acids  =  31.0 kDa
Product: aminoglycoside phosphotransferase
confers resistance to kanamycin
pVS1 StaA
2251 .. 2880  =  630 bp
209 amino acids  =  22.1 kDa
Product: stability protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)
pVS1 StaA
2251 .. 2880  =  630 bp
209 amino acids  =  22.1 kDa
Product: stability protein from the Pseudomonas plasmid pVS1 (Heeb et al., 2000)
ori
12,561 .. 13,149  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
12,561 .. 13,149  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
BlpR
10,332 .. 10,883  =  552 bp
183 amino acids  =  20.6 kDa
Product: phosphinothricin acetyltransferase
confers resistance to bialophos or phosphinothricin
BlpR
10,332 .. 10,883  =  552 bp
183 amino acids  =  20.6 kDa
Product: phosphinothricin acetyltransferase
confers resistance to bialophos or phosphinothricin
35SPPDK promoter
4487 .. 4996  =  510 bp
3 segments
   Segment 1:  CaMV 35S enhancer  
   4487 .. 4785  =  299 bp
hybrid promoter consisting of the cauliflower mosaic virus 35S enhancer fused to the maize C4PPDK basal promoter (Yoo et al., 2007)
35SPPDK promoter
4487 .. 4996  =  510 bp
3 segments
   Segment 2:  
   4786 .. 4808  =  23 bp
hybrid promoter consisting of the cauliflower mosaic virus 35S enhancer fused to the maize C4PPDK basal promoter (Yoo et al., 2007)
35SPPDK promoter
4487 .. 4996  =  510 bp
3 segments
   Segment 3:  C4PPDK basal promoter  
   4809 .. 4996  =  188 bp
hybrid promoter consisting of the cauliflower mosaic virus 35S enhancer fused to the maize C4PPDK basal promoter (Yoo et al., 2007)
35SPPDK promoter
4487 .. 4996  =  510 bp
3 segments
hybrid promoter consisting of the cauliflower mosaic virus 35S enhancer fused to the maize C4PPDK basal promoter (Yoo et al., 2007)
MAS promoter
9946 .. 10,326  =  381 bp
mannopine synthase promoter (Velten et al., 1984)
MAS promoter
9946 .. 10,326  =  381 bp
mannopine synthase promoter (Velten et al., 1984)
MAS terminator
10,893 .. 11,145  =  253 bp
mannopine synthase terminator
MAS terminator
10,893 .. 11,145  =  253 bp
mannopine synthase terminator
NOS terminator
9453 .. 9705  =  253 bp
nopaline synthase terminator and poly(A) signal
NOS terminator
9453 .. 9705  =  253 bp
nopaline synthase terminator and poly(A) signal
pVS1 oriV
489 .. 683  =  195 bp
origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)
pVS1 oriV
489 .. 683  =  195 bp
origin of replication for the Pseudomonas plasmid pVS1 (Heeb et al., 2000)
bom
5 .. 145  =  141 bp
basis of mobility region from pBR322
bom
5 .. 145  =  141 bp
basis of mobility region from pBR322
RB T-DNA repeat
4181 .. 4205  =  25 bp
right border repeat from nopaline C58 T-DNA
RB T-DNA repeat
4181 .. 4205  =  25 bp
right border repeat from nopaline C58 T-DNA
LB T-DNA repeat
11,231 .. 11,255  =  25 bp
left border repeat from nopaline C58 T-DNA
LB T-DNA repeat
11,231 .. 11,255  =  25 bp
left border repeat from nopaline C58 T-DNA
IV2 intron
6544 .. 6732  =  189 bp
modified second intron of the potato ST-LS1 gene (Vancanneyt et al., 1990)
IV2 intron
6544 .. 6732  =  189 bp
modified second intron of the potato ST-LS1 gene (Vancanneyt et al., 1990)
ORF:  3142 .. 3414  =  273 bp
ORF:  90 amino acids  =  9.9 kDa
ORF:  4759 .. 6567  =  1809 bp
ORF:  602 amino acids  =  69.2 kDa
ORF:  6775 .. 9447  =  2673 bp
ORF:  890 amino acids  =  103.1 kDa
ORF:  11,680 .. 12,474  =  795 bp
ORF:  264 amino acids  =  31.0 kDa
ORF:  1382 .. 1954  =  573 bp
ORF:  190 amino acids  =  20.5 kDa
ORF:  2828 .. 3142  =  315 bp
ORF:  104 amino acids  =  12.0 kDa
ORF:  5018 .. 5296  =  279 bp
ORF:  92 amino acids  =  11.1 kDa
ORF:  5642 .. 6067  =  426 bp
ORF:  141 amino acids  =  16.4 kDa
ORF:  7349 .. 7636  =  288 bp
ORF:  95 amino acids  =  11.5 kDa
ORF:  9092 .. 9547  =  456 bp
ORF:  151 amino acids  =  17.4 kDa
ORF:  1719 .. 1976  =  258 bp
ORF:  85 amino acids  =  9.6 kDa
ORF:  3483 .. 3833  =  351 bp
ORF:  116 amino acids  =  12.6 kDa
ORF:  10,332 .. 10,883  =  552 bp
ORF:  183 amino acids  =  20.6 kDa
ORF:  10,935 .. 11,435  =  501 bp
ORF:  166 amino acids  =  18.5 kDa
ORF:  749 .. 1924  =  1176 bp
ORF:  391 amino acids  =  43.9 kDa
ORF:  3179 .. 3865  =  687 bp
ORF:  228 amino acids  =  25.1 kDa
ORF:  10,649 .. 10,981  =  333 bp
ORF:  110 amino acids  =  11.9 kDa
ORF:  2251 .. 2880  =  630 bp
ORF:  209 amino acids  =  22.1 kDa
ORF:  4873 .. 6588  =  1716 bp
ORF:  571 amino acids  =  64.1 kDa
ORF:  10,299 .. 10,808  =  510 bp
ORF:  169 amino acids  =  18.0 kDa
ORF:  12,111 .. 12,341  =  231 bp
ORF:  76 amino acids  =  8.3 kDa
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