pdCas9-bacteria-ApmR vector (Cat. No.: V017644)

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Basic Information

Name:: pdCas9-bacteria-ApmR

Antibiotic Resistance:: Apramycin

Length:: 6818 bp

Type:: Gene-editing vector

Replication origin:: p15A ori

Host:: E. coli

Promoter:: Tet

Growth Strain(s):: Stbl3

Growth Temperature:: 37℃

$ 298.1

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

Resources

Plasmid Protocol

1. Centrifuge at 5,000×g for 5 min.

2. Carefully open the tube and add sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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-ApmR vector (Cat. No.: V017644) Sequence

LOCUS       .                       6818 bp    DNA     circular UNK 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   
VERSION     
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     CDS             95..895
                     /label="ApmR"
                     /note="aminoglycoside 3-N-acetyltransferase type IV"
     terminator      925..1019
                     /label="lambda t0 terminator"
                     /note="transcription terminator from phage lambda"
     rep_origin      1133..1678
                     /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(1840..1867)
                     /label="T7Te terminator"
                     /note="phage T7 early transcription terminator"
     terminator      complement(1883..1954)
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     CDS             complement(1981..6084)
                     /label="dCas9"
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     promoter        complement(6120..6175)
                     /label="tetR/tetA promoters"
                     /note="overlapping promoters for bacterial tetR and tetA"
     CDS             6191..6814
                     /label="TetR"
                     /note="tetracycline repressor TetR"