pKD13 vector (V000040)

Price Information

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

Basic Vector Information

The plasmid pKD13 contains the R6Kγ origin of replication (ori). This particular ori requires the λpir protein for initiation of replication.
For this reason, pKD13 cannot replicate in conventional bacterial strains such as DH5α. DH5α lacks the pir+ gene that is essential for expressing the λpir protein required for pKD13 replication.
However, pKD13 can proliferate in bacteria that contain the pir+ gene, such as S17-1λpir or DH5αλpir. These strains are engineered to express the λpir protein, which enables the replication of pKD13.
It provides specificity in genetic manipulation by only replicating in strains with the pir+ gene. This allows for precise engineering tasks and controlled experiments with reduced background noise.

pKD13 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

• Abdelgader SA, Shi D, Chen M, Zhang L, Hejair HMA, Muhammad U, Yao H, Zhang W. Antibiotics Resistance Genes Screening and Comparative Genomics Analysis of Commensal Escherichia coli Isolated from Poultry Farms between China and Sudan. Biomed Res Int. 2018 Aug 26;2018:5327450.

pKD13 vector Sequence

LOCUS       Exported                3470 bp DNA     circular SYN 31-AUG-2024
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3470)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 3470)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 3470)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..3470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     source          2069..2105
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(51..98)
                     /label=FLP recombinase from the Saccharomyces
                     cerevisi
                     /bound_moiety="FLP recombinase from the Saccharomyces
                     cerevisiae 2u plasmid"
                     /note="FRT"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             complement(114..905)
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
     protein_bind    complement(1273..1306)
                     /label=FRT (minimal)
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     terminator      1386..1632
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     rep_origin      complement(1693..2113)
                     /direction=LEFT
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(2186..3043)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(3044..3148)
                     /label=AmpR promoter
     terminator      3238..3324
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      3416..3443
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"