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Plasmid Files

pFGC-pcoCas9

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

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pFGC-pcoCas9 Sequence and MappFGC-pcoCas9.dna
Map and Sequence File   
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) nucleoplasmin NLS 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

Efficient cleavage requires at least two copies of the XmaI
recognition sequence.
Full cleavage with XmaI may require a long incubation.
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
pcoCas9
5107 .. 9396  =  4290 bp
1367 amino acids  =  158.3 kDa
2 segments
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
   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
   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
Product: Cas9 endonuclease from the Streptococcus
pyogenes
Type II CRISPR/Cas system
plant codon-optimized Cas9 gene containing the
potato IV2 intron
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
   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
   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
   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)
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
MAS terminator
10,893 .. 11,145  =  253 bp
mannopine synthase terminator
MAS terminator
10,893 .. 11,145  =  253 bp
mannopine synthase terminator
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
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
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
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
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
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
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)
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