pKOS6b vector (V005198)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V005198	pKOS6b	In stock, instant shipping	Buy one, get one free!

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

Basic Vector Information

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

Copy Sequence View 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.

pKOS6b vector Sequence

LOCUS       V005198                 6689 bp    DNA     circular SYN 18-DEC-2018
DEFINITION  Exported.
ACCESSION   V005198
VERSION     V005198
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)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl.
            Microbiol. Biotechnol."; date: "2013"; volume: "97"; issue: "18";
            pages: "8341-8349"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (28-SEP-2012) Department of Microbiology, TU Munich, Emil Ramannstr.
            4, Freising, Bayern 85354, Germany"
            SGRef: number: 3; 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"