pdCas9-bacteria vector (V006766)

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Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V006766	pdCas9-bacteria	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:: pdCas9-bacteria

Antibiotic Resistance:: Chloramphenicol

Length:: 6705 bp

Type:: Bacterial Expression, CRISPR

Replication origin:: p15A ori

Copy Number:: Low Copy

Promoter:: pLtetO-1

Cloning Method:: Restriction Enzyme

5' Primer:: ttcaaaaGATCTAAAGAGGAGAAAGGATCTATGGATAAGAAATACTCAATAG

3' Primer:: tgcctggagatccttactcgaGTTAGTCACCTCCTAGCTGACTCAAATCAAT

pdCas9-bacteria 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.

pdCas9-bacteria vector Sequence

LOCUS       pdCas9-bacteria.        6705 bp DNA     circular SYN 13-MAY-2021
DEFINITION  aTc-inducible expression of a catalytically inactive bacterial Cas9 
            (S. pyogenes) for bacterial gene knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    pdCas9-bacteria
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6705)
  AUTHORS   Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim 
            WA
  TITLE     Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific 
            Control of Gene Expression.
  JOURNAL   Cell. 2013 Feb 28;152(5):1173-83. doi: 10.1016/j.cell.2013.02.022.
  PUBMED    23452860
REFERENCE   2  (bases 1 to 6705)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 6705)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.cell.2013.02"; journalName: "Cell"; date: "2013-02-28-
            28"; volume: "152"; issue: "5"; pages: "1173-83"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..6705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(10..633)
                     /label=TetR
                     /note="tetracycline repressor TetR"
     promoter        649..704
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     RBS             722..733
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             740..4843
                     /label=dCas9
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     terminator      4870..4941
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4957..4984
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     primer_bind     complement(5012..5029)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5146..5691)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin."
     terminator      complement(5805..5899)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5923..6579)
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
     promoter        complement(6580..6682)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"