Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017085 | pLV hU6-gRNA(anti-sense) hUbC-VP64-dCas9-VP64-T2A-GFP | 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:
- pLV hU6-gRNA(anti-sense) hUbC-VP64-dCas9-VP64-T2A-GFP
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14994 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Promoter:
- UbC
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
pLV hU6-gRNA(anti-sense) hUbC-VP64-dCas9-VP64-T2A-GFP 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.
pLV hU6-gRNA(anti-sense) hUbC-VP64-dCas9-VP64-T2A-GFP vector Sequence
LOCUS V017085 14994 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V017085
VERSION V017085
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14994)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..14994
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 1..1212
/label="UbC promoter"
/note="human ubiquitin C promoter"
CDS 1292..1315
/label="FLAG"
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 1484..1504
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 1514..5617
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 5882..6595
/label="EGFP"
/note="enhanced GFP"
misc_feature 6669..7257
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 7782..7962
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 7994..8218
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 8264..8692
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 8706..9035
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 9083..9130
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 9149..9520
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 9653..9786
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
promoter complement(9871..9901)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(9916..9937)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(10225..10813)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(10987..11844)
/label="AmpR"
/note="beta-lactamase"
promoter complement(11845..11949)
/label="AmpR promoter"
enhancer 12215..12594
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 12595..12797
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 12812..12992
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 13039..13164
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 13657..13890
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 14075..14119
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
CDS 14268..14309
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 14417..14534
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
protein_bind complement(14578..14611)
/label="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
misc_RNA complement(14622..14697)
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(14722..14962)
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"