Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V001637 | pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro | In stock, 1 week for quality controls |
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-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14900 bp
- Type:
- Lentiviral, CRISPR
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Promoter:
- UbC
pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro 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-Puro vector Sequence
LOCUS V001637 14900 bp DNA circular SYN 21-DEC-2021
DEFINITION Exported.
ACCESSION V001637
VERSION V001637
KEYWORDS pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14900)
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 14900)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14900)
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..14900
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(2..21)
/label="SV40pro-F"
/note="SV40 promoter/origin, forward primer"
primer_bind 248..268
/label="pBABE 3'"
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin complement(277..705)
/direction=LEFT
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
polyA_signal complement(751..975)
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
LTR complement(1007..1187)
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind 1692..1708
/label="pBluescriptKS"
/note="For pBluescript vector"
primer_bind 1693..1709
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
misc_feature complement(1712..2300)
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
protein_bind 2325..2358
/label="loxP"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (ATGTATGC) (Shaw et al., 2021)."
CDS complement(2415..3011)
/label="PuroR"
/note="puromycin N-acetyltransferase"
CDS complement(3012..3065)
/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 complement(3072..3092)
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS complement(3159..3353)
/label="KRAB"
/note="Kruppel-associated box (KRAB) transcriptional
repression domain from the human zinc finger protein ZNF10
(Margolin et al., 1994)"
CDS complement(3387..3407)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS complement(3420..7523)
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS complement(7533..7553)
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS complement(7560..7583)
/label="FLAG"
/note="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
regulatory 7625..7634
/label="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
promoter complement(7663..8874)
/label="UbC promoter"
/note="human ubiquitin C promoter"
promoter 8907..9147
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 9172..9247
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
protein_bind complement(9258..9291)
/label="loxP"
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature complement(9335..9452)
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
CDS complement(9560..9601)
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
CDS complement(9750..9794)
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
misc_feature complement(9979..10212)
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature complement(10705..10830)
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
LTR complement(10877..11057)
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
promoter complement(11072..11274)
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
enhancer complement(11275..11654)
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
primer_bind 11826..11845
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
promoter 11920..12024
/label="AmpR promoter"
CDS 12025..12882
/label="AmpR"
/note="beta-lactamase"
rep_origin 13056..13644
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 13798..13815
/label="L4440"
/note="L4440 vector, forward primer"
protein_bind 13932..13953
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 13968..13998
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 14006..14022
/label="lac operator"
/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)."
primer_bind 14030..14046
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
polyA_signal complement(14083..14216)
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
CDS complement(14349..14720)
/label="BleoR"
/note="antibiotic-binding protein"
promoter complement(14739..14786)
/label="EM7 promoter"
/note="synthetic bacterial promoter"