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Plasmid Files

HBT-pcoCas9

Plasmid for transient expression of plant codon-optimized Cas9 in plant cells. Use together with pUC119‑gRNA.

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HBT-pcoCas9.dna
Map and Sequence File:    Download    Open   
Sequence Author:  Sheen Lab / Addgene #52254
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MauBI (7912) NsiI (7808) AflII (7717) PstI (7687) KflI - PpuMI (6905) BstEII (5201) BfuAI - BspMI (4955) MfeI (4885) XcmI (4581) BlpI (4018) EcoRI (24) AhdI (1273) ZraI (2193) AatII (2195) NdeI (2444) BstAPI (2445) KasI (2495) NarI (2496) SfoI (2497) PluTI (2499) AvaI - BsoBI - PaeR7I - XhoI (2664) BmeT110I (2665) PshAI (2883) BmgBI (2946) EcoRV (2974) PspOMI (3030) ApaI (3034) Eco53kI (3163) BspEI - SacI (3165) BsaAI (3194) BamHI (3234) NcoI (3238) HBT-pcoCas9 7940 bp
MauBI  (7912)
1 site
C G C G C G C G G C G C G C G C
NsiI  (7808)
1 site
A T G C A T T A C G T A
AflII  (7717)
1 site
C T T A A G G A A T T C
PstI  (7687)
1 site
C T G C A G G A C G T C
KflI  (6905)
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.
PpuMI  (6905)
1 site
R G G W C C Y Y C C W G G R

Sticky ends from different PpuMI sites may not be compatible.
BstEII  (5201)
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.
BfuAI  (4955)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BfuAI recognition sequence.
Sticky ends from different BfuAI sites may not be compatible.
BfuAI is typically used at 50°C, but is 50% active at 37°C.
BspMI  (4955)
1 site
A C C T G C ( N ) 4 T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the BspMI recognition sequence.
Sticky ends from different BspMI sites may not be compatible.
MfeI  (4885)
1 site
C A A T T G G T T A A C
XcmI  (4581)
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.
BlpI  (4018)
1 site
G C T N A G C C G A N T C G

Sticky ends from different BlpI sites may not be compatible.
EcoRI  (24)
1 site
G A A T T C C T T A A G
AhdI  (1273)
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.
ZraI  (2193)
1 site
G A C G T C C T G C A G
AatII  (2195)
1 site
G A C G T C C T G C A G
NdeI  (2444)
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.
BstAPI  (2445)
1 site
G C A N N N N N T G C C G T N N N N N A C G

Sticky ends from different BstAPI sites may not be compatible.
KasI  (2495)
1 site
G G C G C C C C G C G G
NarI  (2496)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the NarI recognition sequence.
SfoI  (2497)
1 site
G G C G C C C C G C G G
PluTI  (2499)
1 site
G G C G C C C C G C G G

Efficient cleavage requires at least two copies of the PluTI recognition sequence.
AvaI  (2664)
1 site
C Y C G R G G R G C Y C

Sticky ends from different AvaI sites may not be compatible.
BsoBI  (2664)
1 site
C Y C G R G G R G C Y C

Sticky ends from different BsoBI sites may not be compatible.
BsoBI is typically used at 37°C, but can be used at temperatures up to 65°C.
PaeR7I  (2664)
1 site
C T C G A G G A G C T C

PaeR7I does not recognize the sequence CTCTCGAG.
XhoI  (2664)
1 site
C T C G A G G A G C T C
BmeT110I  (2665)
1 site
C Y C G R G G R G C Y C
PshAI  (2883)
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.
BmgBI  (2946)
1 site
C A C G T C G T G C A G

This recognition sequence is asymmetric, so ligating blunt ends generated by BmgBI will not always regenerate a BmgBI site.
EcoRV  (2974)
1 site
G A T A T C C T A T A G

EcoRV is reportedly more prone than its isoschizomer Eco32I to delete a base after cleavage.
PspOMI  (3030)
1 site
G G G C C C C C C G G G
ApaI  (3034)
1 site
G G G C C C C C C G G G

ApaI can be used between 25°C and 37°C.
Eco53kI  (3163)
1 site
G A G C T C C T C G A G
BspEI  (3165)
1 site
T C C G G A A G G C C T
SacI  (3165)
1 site
G A G C T C C T C G A G
BsaAI  (3194)
1 site
Y A C G T R R T G C A Y
BamHI  (3234)
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.
NcoI  (3238)
1 site
C C A T G G G G T A C C
pcoCas9
3342 .. 7631  =  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
3342 .. 7631  =  4290 bp
1367 amino acids  =  158.3 kDa
   Segment 1:  
   3342 .. 4778  =  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
3342 .. 7631  =  4290 bp
1367 amino acids  =  158.3 kDa
   Segment 2:  
   4968 .. 7631  =  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
3342 .. 7631  =  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
AmpR
1200 .. 2060  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 2:  
   1200 .. 1991  =  792 bp
   263 amino acids  =  28.9 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1200 .. 2060  =  861 bp
286 amino acids  =  31.6 kDa
   Segment 1:  signal sequence  
   1992 .. 2060  =  69 bp
   23 amino acids  =  2.6 kDa
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
AmpR
1200 .. 2060  =  861 bp
286 amino acids  =  31.6 kDa
2 segments
Product: β-lactamase
confers resistance to ampicillin, carbenicillin, and related antibiotics
ori
441 .. 1029  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
441 .. 1029  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
35SPPDK promoter
2722 .. 3231  =  510 bp
   Segment 1:  CaMV 35S enhancer  
   2722 .. 3020  =  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
2722 .. 3231  =  510 bp
   Segment 2:  
   3021 .. 3043  =  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
2722 .. 3231  =  510 bp
   Segment 3:  C4PPDK basal promoter  
   3044 .. 3231  =  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
2722 .. 3231  =  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)
NOS terminator
7688 .. 7940  =  253 bp
nopaline synthase terminator and poly(A) signal
NOS terminator
7688 .. 7940  =  253 bp
nopaline synthase terminator and poly(A) signal
AmpR promoter
2061 .. 2165  =  105 bp
AmpR promoter
2061 .. 2165  =  105 bp
2xFLAG
3243 .. 3290  =  48 bp
16 amino acids  =  2.0 kDa
Product: two tandem FLAG® epitope tags
2xFLAG
3243 .. 3290  =  48 bp
16 amino acids  =  2.0 kDa
Product: two tandem FLAG® epitope tags
nucleoplasmin NLS
7632 .. 7679  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from nucleoplasmin
nucleoplasmin NLS
7632 .. 7679  =  48 bp
16 amino acids  =  1.7 kDa
Product: bipartite nuclear localization signal from nucleoplasmin
SV40 NLS
3297 .. 3317  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
SV40 NLS
3297 .. 3317  =  21 bp
7 amino acids  =  883.1 Da
Product: nuclear localization signal of SV40 large T antigen
ATG
3240 .. 3242  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
ATG
3240 .. 3242  =  3 bp
1 amino acid  =  149.2 Da
Product: start codon
IV2 intron
4779 .. 4967  =  189 bp
modified second intron of the potato ST-LS1 gene (Vancanneyt et al., 1990)
IV2 intron
4779 .. 4967  =  189 bp
modified second intron of the potato ST-LS1 gene (Vancanneyt et al., 1990)
ORF:  1330 .. 1596  =  267 bp
ORF:  88 amino acids  =  9.2 kDa
ORF:  2446 .. 2724  =  279 bp
ORF:  92 amino acids  =  10.9 kDa
ORF:  3253 .. 3531  =  279 bp
ORF:  92 amino acids  =  11.1 kDa
ORF:  3877 .. 4302  =  426 bp
ORF:  141 amino acids  =  16.4 kDa
ORF:  5584 .. 5871  =  288 bp
ORF:  95 amino acids  =  11.5 kDa
ORF:  7327 .. 7782  =  456 bp
ORF:  151 amino acids  =  17.4 kDa
ORF:  2994 .. 4802  =  1809 bp
ORF:  602 amino acids  =  69.2 kDa
ORF:  5010 .. 7682  =  2673 bp
ORF:  890 amino acids  =  103.1 kDa
ORF:  1200 .. 2060  =  861 bp
ORF:  286 amino acids  =  31.6 kDa
ORF:  3108 .. 4823  =  1716 bp
ORF:  571 amino acids  =  64.1 kDa
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