pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP vector (V001403)

Price Information

Cat No. Plasmid Name Availability Add to cart
V001403 pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP 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:
pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP
Antibiotic Resistance:
Ampicillin
Length:
14554 bp
Type:
Mammalian Expression, Lentiviral, CRISPR
Replication origin:
ori
Copy Number:
High Copy
Promoter:
SP6

pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP vector Map

pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP14554 bp7001400210028003500420049005600630070007700840091009800105001120011900126001330014000SP6 promoterpBRrevBampRS-markerpGEX 3'pBRforEcoAmpR promoterAmpRoriL4440SV40 promotersmall t intronSV40 NLSSV40 poly(A) signal3' LTRHIV-1 PsiRREgp41 peptideProtein TatcPPT/CTSSV40 promoterSV40 NLSdCas9HASV40 NLSSV40 NLSGCN4_v4GCN4_v4GCN4_v4GCN4_v4GCN4_v4GCN4_v4GCN4_v4GCN4_v4GCN4_v4GCN4_v4SV40 NLSP2ATagBFPSV40 NLSWPRE

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.

pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP vector Sequence

LOCUS       V001403                14554 bp    DNA     circular SYN 13-MAY-2021
DEFINITION  Exported.
ACCESSION   V001403
VERSION     V001403
KEYWORDS    pHRdSV40-dCas9-10xGCN4_v4-P2A-BFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 14554)
  AUTHORS   Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD
  TITLE     A Protein-Tagging System for Signal Amplification in Gene Expression
            and Fluorescence Imaging.
  JOURNAL   Cell. 2014 Oct 8. pii: S0092-8674(14)01227-6. doi:
            10.1016/j.cell.2014.09.039.
   PUBMED   25307933
REFERENCE   2  (bases 1 to 14554)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 14554)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2014
            Oct 8. pii: S0092-8674(14)01227-6. doi: 10.1016/j.cell.2014.09.039."
            SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..14554
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(102..120)
                     /label="SP6 promoter"
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(410..429)
                     /label="pBRrevBam"
                     /note="pBR322 vectors, tet region, downstream of BamHI,
                     reverse primer"
     primer_bind     complement(643..662)
                     /label="pRS-marker"
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     primer_bind     762..784
                     /label="pGEX 3'"
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(822..840)
                     /label="pBRforEco"
                     /note="pBR322 vectors, upsteam of EcoRI site, forward
                     primer"
     promoter        908..1012
                     /label="AmpR promoter"
     CDS             1013..1870
                     /label="AmpR"
                     /note="beta-lactamase"
     rep_origin      2044..2632
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     primer_bind     2786..2803
                     /label="L4440"
                     /note="L4440 vector, forward primer"
     promoter        2878..3207
                     /label="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     intron          4341..4406
                     /label="small t intron"
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             4536..4556
                     /label="SV40 NLS"
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     polyA_signal    4981..5115
                     /label="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     LTR             5283..5916
                     /label="3' LTR"
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    5963..6088
                     /label="HIV-1 Psi"
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    6585..6818
                     /label="RRE"
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
     CDS             7003..7047
                     /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             7196..7237
                     /note="Protein Tat from Human immunodeficiency virus type 1
                     group M subtype B (isolate WMJ22). Accession#: P12509"
                     /label="Protein Tat"
     misc_feature    7332..7449
                     /label="cPPT/CTS"
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        7512..7722
                     /label="SV40 promoter"
                     /note="SV40 early promoter"
     rep_origin      7581..7716
                     /label="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             7738..7758
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             7813..11916
                     /label="dCas9"
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             11920..11946
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label="HA"
                     /translation="YPYDVPDYA"
     CDS             11965..11985
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             11992..12012
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             12076..12132
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility
                     while preserving binding to an scFv-GFP fusion protein
                     (Tanenbaum et al., 2014)"
                     /label="GCN4_v4"
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             12148..12204
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility
                     while preserving binding to an scFv-GFP fusion protein
                     (Tanenbaum et al., 2014)"
                     /label="GCN4_v4"
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             12220..12276
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility
                     while preserving binding to an scFv-GFP fusion protein
                     (Tanenbaum et al., 2014)"
                     /label="GCN4_v4"
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             12292..12348
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility
                     while preserving binding to an scFv-GFP fusion protein
                     (Tanenbaum et al., 2014)"
                     /label="GCN4_v4"
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             12364..12420
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility
                     while preserving binding to an scFv-GFP fusion protein
                     (Tanenbaum et al., 2014)"
                     /label="GCN4_v4"
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             12436..12492
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility
                     while preserving binding to an scFv-GFP fusion protein
                     (Tanenbaum et al., 2014)"
                     /label="GCN4_v4"
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             12508..12564
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility
                     while preserving binding to an scFv-GFP fusion protein
                     (Tanenbaum et al., 2014)"
                     /label="GCN4_v4"
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             12580..12636
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility
                     while preserving binding to an scFv-GFP fusion protein
                     (Tanenbaum et al., 2014)"
                     /label="GCN4_v4"
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             12652..12708
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility
                     while preserving binding to an scFv-GFP fusion protein
                     (Tanenbaum et al., 2014)"
                     /label="GCN4_v4"
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             12724..12780
                     /codon_start=1
                     /product="GCN4 peptide optimized to improve solubility
                     while preserving binding to an scFv-GFP fusion protein
                     (Tanenbaum et al., 2014)"
                     /label="GCN4_v4"
                     /translation="EELLSKNYHLENEVARLKK"
     CDS             12862..12882
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label="SV40 NLS"
                     /translation="PKKKRKV"
     CDS             12892..12948
                     /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             12961..13659
                     /label="TagBFP"
                     /note="monomeric blue fluorescent protein"
     CDS             13669..13689
                     /label="SV40 NLS"
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     misc_feature    13711..14299
                     /label="WPRE"
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"