pCP20 vector (V001036)

Price Information

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

Basic Vector Information

pCP20 is an ampicillin and CmR plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis. CmR and KmR mutants were transformed with pCP20, and ampicillin-resistant transformants were selected at 30°C, after which a few were colony-purified once nonselectively at 43°C and then tested for loss of all antibiotic resistances. The majority lost the FRT-flanked resistance gene and the FLP helper plasmid simultaneously. This vector was first published in 1995, by Dr. W. Wackernagel, Genetik, Fachbereich Biologic, University Oldenburg.

pCP20 vector Map

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

• Cherepanov PP, Wackernagel W. Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene. 1995 May 26;158(1):9-14. doi: 10.1016/0378-1119(95)00193-a. PMID: 7789817.

pCP20 vector Sequence

LOCUS       V001036                 9332 bp    DNA     circular SYN 13-JAN-2022
DEFINITION  Exported.
ACCESSION   V001036
VERSION     V001036
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 9332)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 9332)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9332
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(564..1220)
                     /label="CmR"
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(1221..1323)
                     /label="cat promoter"
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      1875..2097
                     /label="pSC101 ori"
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             2145..3092
                     /label="Rep101(Ts)"
                     /note="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
     CDS             complement(4016..4360)
                     /gene="mbeC"
                     /label="Mobilization protein MbeC"
                     /note="Mobilization protein MbeC from Escherichia coli.
                     Accession#: P13657"
     CDS             complement(4560..5417)
                     /label="AmpR"
                     /note="beta-lactamase"
     promoter        complement(5418..5522)
                     /label="AmpR promoter"
     CDS             complement(6559..7827)
                     /label="FLP"
                     /note="site-specific recombinase"
     CDS             7928..8638
                     /label="lambda repressor (ts)"
                     /note="temperature-sensitive variant of the phage lambda
                     repressor"