pHR-SFFV-KRAB-dCas9-P2A-mCherry vector (V001281)

COA reports Sequencing Results MSDS Download GenBank File(.gb)

Price Information

Cat No.	Plasmid Name	Availability	Add to cart
V001281	pHR-SFFV-KRAB-dCas9-P2A-mCherry	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:: pHR-SFFV-KRAB-dCas9-P2A-mCherry

Antibiotic Resistance:: Ampicillin

Length:: 14247 bp

Type:: Mammalian Expression, Lentiviral, CRISPR

Replication origin:: ori

Selection Marker:: mCherry

Copy Number:: High Copy

Promoter:: SFFV

Cloning Method:: Gibson Cloning

5' Primer:: gcttcccgagctctataaaagag

3' Primer:: CCAGAGGTTGATTATCGATAAGC

pHR-SFFV-KRAB-dCas9-P2A-mCherry vector Map

Copy Sequence View 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.

pHR-SFFV-KRAB-dCas9-P2A-mCherry vector Sequence

LOCUS       V001281                14247 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V001281
VERSION     V001281
KEYWORDS    pHR-SFFV-KRAB-dCas9-P2A-mCherry
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 14247)
  AUTHORS   Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead
            EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M,
            Weissman JS
  TITLE     Genome-Scale CRISPR-Mediated Control of Gene Repression and
            Activation.
  JOURNAL   Cell. 2014 Oct 23;159(3):647-61. doi: 10.1016/j.cell.2014.09.029.
            Epub 2014 Oct 9.
   PUBMED   25307932
REFERENCE   2  (bases 1 to 14247)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 14247)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; doi:
            "10.1016/j.cell.2014.09.029"; journalName: "Cell"; date:
            "2014-10-23- 23"; volume: "159"; issue: "3"; pages: "647-61"
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..14247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1..17)
                     /label="KS primer"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     LTR             108..341
                     /label="3' LTR (Delta-U3)"
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     promoter        complement(366..384)
                     /label="SP6 promoter"
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(674..693)
                     /label="pBRrevBam"
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
     primer_bind     complement(907..926)
                     /label="pRS-marker"
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     1026..1048
                     /label="pGEX 3'"
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1086..1104)
                     /label="pBRforEco"
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        1172..1276
                     /label="AmpR promoter"
     CDS             1277..2134
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      2308..2896
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     primer_bind     3050..3067
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     promoter        3142..3471
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     intron          4605..4670
                     /label="small t intron"
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4800..4820
                     /label="SV40 NLS"
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     polyA_signal    5245..5379
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     LTR             5547..6180
                     /label="3' LTR"
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    6227..6352
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    6849..7082
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     CDS             7267..7311
                     /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             7460..7501
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     misc_feature    7596..7713
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        7866..8273
                     /label="SFFV promoter"
                     /note="spleen focus-forming virus long terminal repeat
                     (LTR) promoter"
     regulatory      8348..8357
                     /label="Kozak sequence"
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             8384..8569
                     /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="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEP"
     CDS             8591..12694
                     /label="dCas9"
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             12698..12724
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label="HA"
                     /translation="YPYDVPDYA"
     CDS             12743..12763
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             12770..12790
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             12830..12886
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /label="P2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             12887..13594
                     /label="mCherry"
                     /note="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
     misc_feature    13657..14245
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"