pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro vector (V001637)

Price Information

Cat No. Plasmid Name Availability Add to cart
V001637 pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro In stock, 1 week for quality controls

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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

pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-Puro14900 bp700140021002800350042004900560063007000770084009100980010500112001190012600133001400014700SV40pro-FpBABE 3'f1 oribGH poly(A) signal5' LTR (truncated)KS primerWPREloxPPuroRT2ASV40 NLSKRABSV40 NLSdCas9SV40 NLSFLAGKozak sequenceUbC promoterU6 promotergRNA scaffoldloxPcPPT/CTSProtein Tatgp41 peptideRREHIV-1 Psi5' LTR (truncated)CMV promoterCMV enhancerpRS-markerAmpR promoterAmpRoriL4440CAP binding sitelac promoterlac operatorM13 revSV40 poly(A) signalBleoREM7 promoter

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"