Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V005198 | pKOS6b | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pKOS6b is a plasmid for markerless gene deletion (via codBA counter-selection with 5-FC) and chromosomal gene insertion (e.g., fluorescent reporters). It enables seamless genetic manipulation of cellulose synthase genes (bcsAB1/2/3) in K. hansenii, with kanamycin resistance and temperature-sensitive replication for plasmid curing.
- Vector Name:
- pKOS6b
- Antibiotic Resistance:
- Kanamycin
- Length:
- 6689 bp
- Type:
- Cloning vector
- Replication origin:
- ori
- Source/Author:
- Kostner D, Peters B, Mientus M, Liebl W, Ehrenreich A.
- Growth Strain(s):
- DH10b
- Growth Temperature:
- 37℃
pKOS6b 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
- Bimmer M, Mientus M,Klingl A, Ehrenreich A, Liebl W. 2022. The Roles of the Various Cellulose Biosynthesis Operons in Komagataeibacter hansenii ATCC 23769. Appl Environ Microbiol 88:e02460-21.
- https://doi.org/10.1128/aem.02460-21
pKOS6b vector Sequence
LOCUS Exported 6689 bp DNA circular SYN 19-JUL-2025
DEFINITION Exported.
ACCESSION V005198
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6689)
AUTHORS Kostner D, Peters B, Mientus M, Liebl W, Ehrenreich A.
TITLE Importance of codB for new codA-based markerless gene deletion in
Gluconobacter strains
JOURNAL Appl. Microbiol. Biotechnol. 97 (18), 8341-8349 (2013)
PUBMED 23955475
REFERENCE 2 (bases 1 to 6689)
AUTHORS Kostner D, Liebl W, Ehrenreich A.
TITLE Direct Submission
JOURNAL Submitted (28-SEP-2012) Department of Microbiology, TU Munich, Emil
Ramannstr. 4, Freising, Bayern 85354, Germany
REFERENCE 3 (bases 1 to 6689)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 6689)
TITLE Direct Submission
REFERENCE 5 (bases 1 to 6689)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Appl.
Microbiol. Biotechnol."; date: "2013"; volume: "97"; issue: "18"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(28-SEP-2012) Department of Microbiology, TU Munich, Emil Ramannstr.
4, Freising, Bayern 85354, Germany"
COMMENT SGRef: number: 3; type: "Journal Article"
COMMENT SGRef: number: 4; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..6689
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 401..1192
/label=NeoR/KanR
/note="aminoglycoside phosphotransferase"
CDS 1495..2751
/gene="codB"
/label=Cytosine permease
/note="Cytosine permease from Escherichia coli (strain
K12). Accession#: P0AA82"
CDS 2744..4024
/label=codA
/note="E. coli cytosine deaminase"
oriT complement(4432..4541)
/direction=LEFT
/label=oriT
/note="incP origin of transfer"
promoter complement(4776..4866)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
promoter complement(5060..5150)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
rep_origin 5510..6098
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 6386..6407
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 6422..6452
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6460..6476
/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)."
primer_bind 6484..6500
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
misc_feature 6509..6565
/label=MCS
/note="pUC18/19 multiple cloning site"
primer_bind complement(6569..6585)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"