HBT-pcoCas9 vector (V012338)

Price Information

Cat No. Plasmid Name Availability Add to cart
V012338 HBT-pcoCas9 In stock, 1 week for quality controls

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

Basic Vector Information

Vector Name:
HBT-pcoCas9
Antibiotic Resistance:
Ampicillin
Length:
7940 bp
Type:
CRISPR Plasmids
Replication origin:
ori
Source/Author:
Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM,
Copy Number:
High copy number
Promoter:
35SPPDK

HBT-pcoCas9 vector Map

HBT-pcoCas97940 bp30060090012001500180021002400270030003300360039004200450048005100540057006000630066006900720075007800M13 revlac operatorlac promoterCAP binding siteoriAmpRAmpR promoterM13 fwd35SPPDK promoterATG2xFLAGSV40 NLSIV2 intronCas9(C)nucleoplasmin NLSNOS terminator

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.

3. Close the tube and incubate for 10 minutes at room temperature.

4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.

5. Store the plasmid at -20 ℃.

6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it

General Plasmid Transform Protocol

1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.

2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.

3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.

4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.

5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.

HBT-pcoCas9 vector Sequence

LOCUS       HBT-pcoCas9.        7940 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Plasmid for transient expression of plant codon-optimized Cas9 in 
            plant cells. Use together with pUC119-gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    HBT-pcoCas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7940)
  AUTHORS   Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM,
            Sheen J.
  TITLE     Multiplex and homologous recombination-mediated genome editing in 
            Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.
  JOURNAL   Nat. Biotechnol. 2013;31:688-91.
  PUBMED    23929339
REFERENCE   2  (bases 1 to 7940)
  AUTHORS   Sheen Lab / Addgene #52254
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7940)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat. 
            Biotechnol."; date: "2013"; volume: "31"; pages: "688-91"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7940
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..55)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(63..79)
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(87..117)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(132..153)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(441..1029)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1203..2060)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(2061..2165)
                     /label=AmpR promoter
     primer_bind     2639..2655
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2722..3231
                     /label=35SPPDK promoter
                     /note="hybrid promoter consisting of the cauliflower mosaic
                     virus 35S enhancer fused to the maize C4PPDK basal promoter
                     (Yoo et al., 2007)"
     CDS             3240..3242
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             3243..3290
                     /label=2xFLAG
                     /note="two tandem FLAG(R) epitope tags"
     CDS             3297..3317
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     intron          4779..4967
                     /label=IV2 intron
                     /note="modified second intron of the potato ST-LS1 gene 
                     (Vancanneyt et al., 1990)"
     CDS             5133..7631
                     /label=Cas9(C)
                     /note="C-terminal portion of Streptococcus pyogenes Cas9 
                     (Zetsche et al., 2015)"
     CDS             7632..7679
                     /label=nucleoplasmin NLS
                     /note="bipartite nuclear localization signal from
                     nucleoplasmin"
     terminator      7688..7940
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"