Cart 2

pThermoCas9_ctrl vector (V007240)

Price Information

Cat No. Plasmid Name Availability Add to cart
V007240 pThermoCas9_ctrl 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:
pThermoCas9_ctrl
Antibiotic Resistance:
Chloramphenicol
Length:
7467 bp
Type:
Bacterial Expression
Replication origin:
ori
Promoter:
B. coagulans DSM 1 pta promoter
Cloning Method:
Gibson Cloning
5' Primer:
ggatcccatgacgctagtatccagctggGTCATAGTTCCCCTGAGAT
3' Primer:
CCCTCCCATGCACAATAG

pThermoCas9_ctrl vector Map

Copy Sequence View Map
pThermoCas9_ctrl7467 bp300600900120015001800210024002700300033003600390042004500480051005400570060006300660069007200M13 revlac operatorlac promoterCAP binding siteChloramphenicol acetyltransferaserepBL4440ori

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.

pThermoCas9_ctrl vector Sequence

LOCUS       V007240                 7467 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V007240
VERSION     V007240
KEYWORDS    pThermoCas9_ctrl
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 7467)
  AUTHORS   Mougiakos I, Mohanraju P, Bosma EF, Vrouwe V, Finger Bou M,
            Naduthodi MIS, Gussak A, Brinkman RBL, van Kranenburg R, van der
            Oost J
  TITLE     Characterizing a thermostable Cas9 for bacterial genome editing and
            silencing.
  JOURNAL   Nat Commun. 2017 Nov 21;8(1):1647. doi: 10.1038/s41467-017-01591-4.
   PUBMED   29162801
REFERENCE   2  (bases 1 to 7467)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 7467)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat
            Commun."; date: "2017-11-21"; pages: "
            10.1038/s41467-017-01591-4"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7467
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(46..62)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(70..86)
                     /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(94..124)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(139..160)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     CDS             complement(293..940)
                     /gene="cat"
                     /label="Chloramphenicol acetyltransferase"
                     /note="Chloramphenicol acetyltransferase from
                     Staphylococcus aureus. Accession#: P00485"
     CDS             complement(1050..2051)
                     /label="repB"
                     /note="RepB replication protein"
     primer_bind     complement(2572..2589)
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2743..3331)
                     /direction=LEFT
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"