Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V012920 | pKC1139 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
The plasmid pKC1139 is a temperature-sensitive, gene-disruption vector primarily used for targeted gene knockout via homologous recombination in Streptomyces and other actinomycetes. It features an apramycin resistance marker for selection and a temperature-sensitive replicon that facilitates genetic manipulation.
- Vector Name:
- pKC1139
- Antibiotic Resistance:
- Apramycin
- Length:
- 6708 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- E. coli
- Growth Strain(s):
- DH10B
- Growth Temperature:
- 30℃
pKC1139 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
- Bridget AF, Budhathoki R, Huo C, Joshi S, Parajuli N, Sohng JK, Kim KH. Activation of cryptic biosynthetic pathways in Saccharopolyspora spinosa through deletion of the spinosyn gene cluster: induction of cryptic and bioactive natural products. Arch Pharm Res. 2025 Jun;48(6):514-527. doi: 10.1007/s12272-025-01553-1. Epub 2025 Jun 17. PMID: 40528115.
- Schaffert L, März C, Burkhardt L, Droste J, Brandt D, Busche T, Rosen W, Schneiker-Bekel S, Persicke M, Pühler A, Kalinowski J. Evaluation of vector systems and promoters for overexpression of the acarbose biosynthesis gene acbC in Actinoplanes sp. SE50/110. Microb Cell Fact. 2019 Jun 28;18(1):114. doi: 10.1186/s12934-019-1162-5. PMID: 31253141; PMCID: PMC6599336.
pKC1139 vector Sequence
LOCUS Exported 6708 bp DNA circular SYN 15-JUL-2025
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6708)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 6708)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6708
/mol_type="other DNA"
/organism="synthetic DNA construct"
source 6162..6297
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS complement(170..508)
/codon_start=1
/gene="lacZ fragment"
/product="LacZ-alpha fragment of beta-galactosidase"
/label=lacZ-alpha
/translation="MITNSISRAAADPLESTCSPSLALAVVLQRRDWENPGVTQLNRLA
AHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRINSPMSSTSGIG
SAADAKCR"
primer_bind 424..440
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(509..525)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
protein_bind complement(533..549)
/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(557..587)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind complement(602..623)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(911..1499)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS 1703..2503
/codon_start=1
/label=ApmR
/note="aminoglycoside 3-N-acetyltransferase type IV"
/translation="MSSAVECNVVQYEWRKAELIGQLLNLGVTPGGVLLVHSSFRSVRP
LEDGPLGLIEALRAALGPGGTLVMPSWSGLDDEPFDPATSPVTPDLGVVSDTFWRLPNV
KRSAHPFAFAAAGPQAEQIISDPLPLPPHSPASPVARVHELDGQVLLLGVGHDANTTLH
LAELMAKVPYGVPRHCTILQDGKLVRVDYLENDHCCERFALADRWLKEKSLQKEGPVGH
AFARLIRSRDIVATALGQLGRDPLIFLHPPEAGCEECDAARQSIG"
CDS complement(3442..4884)
/codon_start=1
/gene="rep"
/product="replication initiator protein from the
Streptomyces ghanaensis plasmid pSG5"
/label=pSG5 Rep
/translation="MWKPGEATWGNTCRCNNVHTCPWCMSRILAVRGSNVQLAADGLAD
AGYGLHLGTNTLRHFERMAFGTVRKGMRHGLVAVLHDGWKGAYGSSGRRWRTMRDDFGI
IGYERAFEDTFGWGSGWHLHWHTLWVTREVLGPDAQAAFRDALAGAWAAGVESAGGYTV
SETCDRPGCSCEGKGHGTDVRPLNGADAADGDAGKQARYLYKDGDKTKGGVAKIGLELA
GQNFKAGRGDDRMGPLDLGDAAAAELQRLRRPGPFVEKYREREFGVFQVRKHYRSQNLN
RLIKELGIQQDVRTEEEITDDTEGLVAIAVIPAYIWYRYIARVAGRRLDLIKVAETYGL
PGVRRLVESWGLVWGKDVLDPPAPEAPAAPGDLDADQMRFEVMSEEEAAFREARRKANE
ARTEELAASLDRVRQPKKEAIRPTISLRKRLKPKPVTVDVKTPPPGAASPVCRRCKGKL
APVLQPWGQHPGDCLRVDTAVA"
oriT 6051..6160
/label=oriT
/note="incP origin of transfer"
CDS 6193..6564
/codon_start=1
/gene="traJ"
/product="oriT-recognizing protein"
/label=traJ
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"