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pCB578 vector (V017331)

Price Information

Cat No. Plasmid Name Availability Add to cart
V017331 pCB578 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:
pCB578
Antibiotic Resistance:
Erythromycin
Length:
11446 bp
Type:
Genome editing
Replication origin:
ori
Host:
Lactobacillus
Copy Number:
Low
Growth Temperature:
37℃

pCB578 vector Vector Map

pCB57811446 bp500100015002000250030003500400045005000550060006500700075008000850090009500100001050011000tracrRNACas9oriT7 promoterDRrRNA adenine N-6-methyltransferase

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.

pCB578 vector Sequence

Copy Sequence

Download GeneBank File(.gb)

LOCUS       V017331                11446 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V017331
VERSION     V017331
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 11446)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..11446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        complement(3024..3102)
                     /label="tracrRNA"
                     /note="trans-activating CRISPR RNA for the Streptococcus
                     pyogenes CRISPR/Cas9 system"
     CDS             3398..7501
                     /label="Cas9"
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     rep_origin      8046..8634
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     promoter        8892..8910
                     /label="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   9479..9514
                     /label="DR"
                     /note="direct repeat for the Streptococcus pyogenes
                     CRISPR/Cas system"
     CDS             10199..10933
                     /gene="ermBP"
                     /label="rRNA adenine N-6-methyltransferase"
                     /note="rRNA adenine N-6-methyltransferase from Enterococcus
                     faecalis. Accession#: P0A4D5"