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pCB578 vector (V017331) Gene synthesis in pCB578 backbone

Price Information

Cat No. Plasmid Name Availability Buy one, get one free! (?)
V017331 pCB578 In stock, instant shipping

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

Basic Vector Information

E. coli-Lactobacilli shuttle vector featuring SpCas9, tracrRNA, and a repeat-spacer-repeat array, with erythromycin resistance at 200 μg/mL.

Vector Name:
pCB578
Antibiotic Resistance:
Erythromycin
Length:
11446 bp
Type:
Genome editing
Replication origin:
ori
Host:
Lactobacillus
Copy Number:
Low
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

pCB578 vector Map

Copy Sequence View Map
pCB57811446 bp500100015002000250030003500400045005000550060006500700075008000850090009500100001050011000copSrepDRepRtracrRNACas9oriT7 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.

References

  • Leenay RT, Vento JM, Shah M, Martino ME, Leulier F, Beisel CL. Genome Editing with CRISPR-Cas9 in Lactobacillus plantarum Revealed That Editing Outcomes Can Vary Across Strains and Between Methods. Biotechnol J. 2019;14(3):e1700583. doi:10.1002/biot.201700583

pCB578 vector Sequence

LOCUS       Exported               11446 bp DNA     circular SYN 13-SEP-2024
DEFINITION  Exported.
ACCESSION   V017331
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11446)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 11446)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..11446
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          279..563
                     /mol_type="other DNA"
                     /label=broad host range vector
                     /note="broad host range vector"
                     /db_xref="taxon:1265608"
                     /organism="Shuttle vector pKV12"
     source          join(564..11446,1..278)
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          899..1228
                     /mol_type="other DNA"
                     /label=broad host range vector
                     /note="broad host range vector"
                     /db_xref="taxon:1265608"
                     /organism="Shuttle vector pKV12"
     gene            279..563
                     /gene="copS"
                     /label=copS
     CDS             279..563
                     /codon_start=1
                     /transl_table=11
                     /gene="copS"
                     /product="copy number repressor"
                     /label=copS
                     /protein_id="AFY24252.1"
                     /translation="MELAFRESLKKMRGTKSKEKFSQELEMSRSNYSRIESGKSDPTIK
                     TLEQIAKLTNSTLVVDLIPNEPTEPEPETESEQVTLDLEMEEEKSNDFV"
     gene            899..1228
                     /gene="repD"
                     /label=repD
     CDS             899..1228
                     /codon_start=1
                     /transl_table=11
                     /gene="repD"
                     /product="replication protein RepD"
                     /label=repD
                     /protein_id="AFY24251.1"
                     /translation="MYSFIEGGVCMNHDECKTYIKNSLLEIRKLANIYTLETFKKELEK
                     RNIYLETKSDKYFSSEGEDYIYKLIENNKIIYSISGKKLTYKGKKSFSKHAILKQLNEK
                     ANQVN"
     CDS             1271..2761
                     /codon_start=1
                     /label=RepR
                     /translation="MNIPFVVETVLHDGLLKYKFKNSKIRSITTKPGKSKGAIFAYRSK
                     SSMIGGRGVVLTSEEAIQENQDTFTHWTPNVYRYGTYADENRSYTKGHSENNLRQINTF
                     FIDFDIHTAKETISASDILTTAIDLGFMPTMIIKSDKGYQAYFVLETPVYVTSKSEFKS
                     VKAAKIISQNIREYFGKSLPVDLTCNHFGIARIPRTDNVEFFDPNYRYSFKEWQDWSFK
                     QTDNKGLTRSSLTVLSGTEGKKQVDEPWFNLLLHETKFSGEKGLIGRNNVMFTLSLAYF
                     SSGYSIETCEYNMFEFNNRLDQPLEEKEVIKIVRSAYSENYQGANREYITILCKAWVSS
                     DLTSKDLFVRQGWFKFKKKRSERQRVHLSEWKEDLMAYISEKSDVYKPYLVTTKKEIRE
                     VLGIPERTLDKLLKVLKANQEIFFKIKPGRNGGIQLASVKSLLLSIIKVKKEEKESYIK
                     ALTNSFDLEHTFIQETLNKLAERPKTDTQLDLFSYDTG"
     misc_RNA        complement(3487..3565)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     CDS             3861..7964
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     rep_origin      8509..9097
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     promoter        9355..9373
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   9942..9977
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes
                     CRISPR/Cas system"
     CDS             10662..11396
                     /gene="ermBP"
                     /label=rRNA adenine N-6-methyltransferase
                     /note="rRNA adenine N-6-methyltransferase from Enterococcus
                     faecalis. Accession#: P0A4D5"