Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001427 | pLV hU6-sgRNA hUbC-dCas9-KRAB-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-sgRNA hUbC-dCas9-KRAB-T2a-GFP
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14982 bp
- Type:
- Lentiviral, CRISPR
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- UbC
pLV hU6-sgRNA hUbC-dCas9-KRAB-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-sgRNA hUbC-dCas9-KRAB-T2a-GFP vector Sequence
LOCUS V001427 14982 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V001427
VERSION V001427
KEYWORDS pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14982)
AUTHORS Thakore PI, D'Ippolito AM, Song L, Safi A, Shivakumar NK, Kabadi AM,
Reddy TE, Crawford GE, Gersbach CA
TITLE Highly specific epigenome editing by CRISPR-Cas9 repressors for
silencing of distal regulatory elements.
JOURNAL Nat Methods. 2015 Dec;12(12):1143-9. doi: 10.1038/nmeth.3630. Epub
2015 Oct 26.
PUBMED 26501517
REFERENCE 2 (bases 1 to 14982)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14982)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1038/nmeth.3630"; journalName: "Nat Methods"; date: "2015-12";
volume: "12"; issue: "12"; pages: "1143-9"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14982
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 1..380
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 381..583
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 598..778
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 825..950
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1443..1676
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1861..1905
/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 2054..2095
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2203..2320
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
protein_bind complement(2364..2397)
/label="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
misc_RNA complement(2408..2483)
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter complement(2508..2748)
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
promoter 2781..3992
/label="UbC promoter"
/note="human ubiquitin C promoter"
regulatory 4021..4030
/label="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 4072..4095
/label="FLAG"
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
CDS 4102..4122
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 4132..8235
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 8248..8268
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 8302..8496
/codon_start=1
/product="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
/label="KRAB"
/translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
QLTKPDVILRLEKGEEPWLV"
CDS 8563..8583
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 8590..8643
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 8650..9363
/label="EGFP"
/note="enhanced GFP"
protein_bind complement(9379..9412)
/label="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
primer_bind complement(9490..9510)
/label="WPRE-R"
/note="WPRE, reverse primer"
CDS complement(9908..9919)
/label="Factor Xa site"
/note="Factor Xa recognition and cleavage site"
primer_bind complement(10028..10044)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10029..10045)
/label="pBluescriptKS"
/note="For pBluescript vector"
LTR 10550..10730
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 10762..10986
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 11032..11460
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 11474..11803
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 11851..11898
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 11917..12288
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 12421..12554
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(12591..12607)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12591..12607)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12604..12626)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 12615..12631
/label="lac operator"
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(12639..12669)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(12684..12705)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12822..12839)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(12993..13581)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13755..14612)
/label="AmpR"
/note="beta-lactamase"
promoter complement(14613..14717)
/label="AmpR promoter"
primer_bind complement(14792..14811)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"