pJK02 vector (V000706)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V000706	pJK02	In stock, 1 week for quality controls	Buy one, get one free!

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

Basic Vector Information

Vector Name:: pJK02

Antibiotic Resistance:: Chloramphenicol

Length:: 14173 bp

Type:: E. coli - C. difficile shuttle vector

Replication origin:: ori

Copy Number:: Low Copy

pJK02 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.

pJK02 vector Sequence

LOCUS       V000706                14173 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V000706
VERSION     V000706
KEYWORDS    pJK02
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 14173)
  AUTHORS   McAllister KN, Bouillaut L, Kahn JN, Self WT, Sorg JA
  TITLE     Using CRISPR-Cas9-mediated genome editing to generate C. difficile
            mutants defective in selenoproteins synthesis.
  JOURNAL   Sci Rep. 2017 Nov 7;7(1):14672. doi: 10.1038/s41598-017-15236-5.
   PUBMED   29116155
REFERENCE   2  (bases 1 to 14173)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 14173)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep.";
            date: "2017-11-7"; pages: "
            10.1038/s41598-017-15236-5"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..14173
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     63..79
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     primer_bind     63..79
                     /label="M13 Reverse"
                     /note="In lacZ gene. Also called M13-rev"
     CDS             102..1001
                     /gene="pyrD"
                     /label="Dihydroorotate dehydrogenase B (NAD(+)), catalytic
                     subunit"
                     /note="Dihydroorotate dehydrogenase B (NAD(+)), catalytic
                     subunit from Clostridioides difficile (strain 630).
                     Accession#: Q18CS6"
     CDS             complement(2117..2740)
                     /label="TetR"
                     /note="tetracycline repressor TetR"
     protein_bind    2847..2865
                     /label="tet operator"
                     /note="bacterial operator O1 for the tetR and tetA genes"
     CDS             2938..7041
                     /label="Cas9"
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     primer_bind     complement(7054..7071)
                     /label="M13 Forward"
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21)
                     Forward"
     primer_bind     complement(7054..7070)
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     primer_bind     complement(7063..7085)
                     /label="M13/pUC Forward"
                     /note="In lacZ gene"
     CDS             10796..11416
                     /gene="catP"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from Clostridium
                     perfringens. Accession#: P26826"
     misc_RNA        12344..12419
                     /label="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     rep_origin      12617..13205
                     /direction=RIGHT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     primer_bind     13359..13376
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     oriT            13526..13635
                     /label="oriT"
                     /note="incP origin of transfer"
     CDS             13668..14036
                     /label="traJ"
                     /note="oriT-recognizing protein"