BB3cH_pGAP_23*_pLAT1_Cas9 vector (V017633) Gene synthesis in BB3cH_pGAP_23*_pLAT1_Cas9 backbone

Price Information

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

Basic Vector Information

BB3cH_pGAP_23*pLAT1_Cas9 is a multiple genome editing vector for Pichia pastoris, providing constitutive sgRNA expression and nitrogen-source-regulated Cas9 expression to achieve one-step multi-gene knockout/integration.

BB3cH_pGAP_23*_pLAT1_Cas9 vector 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.

References

• Gassler T, Heistinger L, Mattanovich D, Gasser B, Prielhofer R. CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris. Methods Mol Biol. 2019;1923:211-225. doi: 10.1007/978-1-4939-9024-5_9. PMID: 30737742.

BB3cH_pGAP_23*_pLAT1_Cas9 vector Sequence

LOCUS       .                       9323 bp    DNA     circular UNK 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   
VERSION     
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     rep_origin      complement(62..650)
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     promoter        737..1213
                     /label="GAP promoter"
                     /note="promoter for Pichia pastoris
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             2757..6860
                     /label="Cas9"
                     /note="S. pyogenes Cas9, codon optimized for C. elegans
                     with synthetic introns"
     terminator      6965..7212
                     /label="CYC1 terminator"
                     /note="transcription terminator for CYC1"
     gene            7232..8807
                     /label="hphMX6"
                     /note="yeast selectable marker conferring hygromycin
                     resistance"
     misc_feature    8819..9322
                     /label="CEN/ARS"
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"