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PiggyBac-U6-MCS-sgRNA-Cas9-Puro vector (V017970) Gene synthesis in PiggyBac-U6-MCS-sgRNA-Cas9-Puro backbone

Price Information

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V017970 PiggyBac-U6-MCS-sgRNA-Cas9-Puro In stock, instant shipping

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

Basic Vector Information

This vector mediates CRISPRi/a in human pluripotent stem cells via PiggyBac (stable integration), U6-driven sgRNA (MCS for targets), Cas9/dCas9 regulation, and Puro selection, supporting multi-gene control.

Vector Name:
PiggyBac-U6-MCS-sgRNA-Cas9-Puro
Antibiotic Resistance:
Ampicillin
Length:
12154 bp
Type:
Gene-editing vector
Replication origin:
ori
Host:
Mammalian cells
Selection Marker:
Puro
Promoter:
CMV
3' Primer:
Sv40-polyA-R
Growth Strain(s):
DH10B
Growth Temperature:
37℃

PiggyBac-U6-MCS-sgRNA-Cas9-Puro vector Map

Copy Sequence View Map
PiggyBac-U6-MCS-sgRNA-Cas9-Puro12154 bp60012001800240030003600420048005400600066007200780084009000960010200108001140012000AmpR promoterf1 oriU6 promotergRNA scaffoldEF-1α core promoterCas9FLAGP2APuroRSV40 poly(A) signallac promoterCAP binding siteoriAmpR

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

  • Hazelbaker, D.Z., Beccard, A., Angelini, G. et al. A multiplexed gRNA piggyBac transposon system facilitates efficient induction of CRISPRi and CRISPRa in human pluripotent stem cells. Sci Rep 10, 635 (2020). https://doi.org/10.1038/s41598-020-57500-1

PiggyBac-U6-MCS-sgRNA-Cas9-Puro vector Sequence

LOCUS       .                      12154 bp    DNA     circular UNK 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   
VERSION     
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     promoter        complement(1..105)
                     /label="AmpR promoter"
     rep_origin      complement(131..586)
                     /label="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        1365..1605
                     /label="U6 promoter"
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_RNA        3495..3570
                     /label="gRNA scaffold"
                     /note="guide RNA scaffold for the Streptococcus pyogenes
                     CRISPR/Cas9 system"
     promoter        3632..3843
                     /label="EF-1α core promoter"
                     /note="core promoter for human elongation factor EF-1α"
     CDS             3868..7971
                     /label="Cas9"
                     /note="S. pyogenes Cas9, codon optimized for C. elegans
                     with synthetic introns"
     CDS             8020..8043
                     /label="FLAG"
                     /note="FLAG® epitope tag, followed by an enterokinase
                     cleavage site"
     CDS             8053..8109
                     /label="P2A"
                     /note="2A peptide from porcine teschovirus-1 polyprotein"
     CDS             8110..8703
                     /label="PuroR"
                     /note="puromycin N-acetyltransferase"
     polyA_signal    8725..8857
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     promoter        complement(10181..10211)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(10226..10247)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(10535..11123)
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     CDS             complement(11297..12154)
                     /label="AmpR"
                     /note="β-lactamase"