Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017087 | pLentiRNACRISPR_006 - hU6-DR_BsmBI-EFS-RfxCas13d-NES-2A-Puro-WPRE | 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:
- pLentiRNACRISPR_006 - hU6-DR_BsmBI-EFS-RfxCas13d-NES-2A-Puro-WPRE
- Antibiotic Resistance:
- Ampicillin
- Length:
- 11728 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro
- Promoter:
- U6
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
pLentiRNACRISPR_006 - hU6-DR_BsmBI-EFS-RfxCas13d-NES-2A-Puro-WPRE vector 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.
pLentiRNACRISPR_006 - hU6-DR_BsmBI-EFS-RfxCas13d-NES-2A-Puro-WPRE vector Sequence
LOCUS V017087 11728 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V017087
VERSION V017087
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 11728)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..11728
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 249..628
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 629..831
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 846..1026
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1073..1198
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1691..1924
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2109..2153
/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 2302..2343
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2451..2568
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2619..2859
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 2869..2898
/label="RfxCas13d DR30"
/note="5' processed 30-base direct repeat for an RfxCas13d
gRNA (Konermann et al., 2018)"
promoter 2977..3188
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 3213..6110
/label="RfxCas13d"
/note="RNA-targeting Type VI CRISPR protein from
Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
2018)"
CDS 6117..6143
/label="NES"
/note="nuclear export signal from the HIV Rev protein
(Fischer et al., 1995)"
CDS 6162..6218
/label="P2A"
/note="2A peptide from porcine teschovirus-1 polyprotein"
CDS 6219..6812
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 6831..7419
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 7491..7724
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 7756..7980
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 8026..8454
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 8468..8797
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 8845..8892
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 8911..9282
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 9415..9548
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
promoter complement(9633..9663)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(9678..9699)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
rep_origin complement(9987..10575)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(10749..11606)
/label="AmpR"
/note="beta-lactamase"
promoter complement(11607..11711)
/label="AmpR promoter"