Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001133 | pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE | 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:
- pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14914 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- SV40
- Cloning Method:
- Restriction Enzyme
- 5' Primer:
- SV40pro-F2
- 3' Primer:
- mTagBFP-R
pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE 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.
pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE vector Sequence
LOCUS V001133 14914 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V001133
VERSION V001133
KEYWORDS pHRdSV40-NLS-dCas9-24xGCN4_v4-NLS-P2A-BFP-dWPRE
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14914)
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 14914)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14914)
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..14914
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 90..419
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
intron 1553..1618
/label="small t intron"
/note="SV40 (simian virus 40) small t antigen intron"
CDS 1748..1768
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
polyA_signal 2193..2327
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
LTR 2495..3128
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
misc_feature 3175..3300
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 3797..4030
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 4215..4259
/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 4408..4449
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 4544..4661
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 4724..4934
/label="SV40 promoter"
/note="SV40 early promoter"
rep_origin 4793..4928
/label="SV40 ori"
/note="SV40 origin of replication"
CDS 4950..4970
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 5025..9128
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 9132..9158
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label="HA"
/translation="YPYDVPDYA"
CDS 9177..9197
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 9204..9224
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 9303..9359
/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 9375..9431
/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 9447..9503
/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 9519..9575
/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 9591..9647
/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 9663..9719
/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 9735..9791
/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 9807..9863
/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 9879..9935
/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 9951..10007
/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 10023..10079
/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 10095..10151
/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 10167..10223
/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 10239..10295
/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 10311..10367
/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 10383..10439
/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 10455..10511
/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 10527..10583
/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 10599..10655
/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 10671..10727
/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 10743..10799
/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 10815..10871
/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 10959..11015
/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 11055..11075
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS 11085..11141
/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 11154..11852
/label="TagBFP"
/note="monomeric blue fluorescent protein"
LTR 11970..12203
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
promoter complement(12228..12246)
/label="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(12536..12555)
/label="pBRrevBam"
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind complement(12769..12788)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 12888..12910
/label="pGEX 3'"
/note="pGEX vectors, reverse primer"
primer_bind complement(12948..12966)
/label="pBRforEco"
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 13034..13138
/label="AmpR promoter"
CDS 13139..13996
/label="AmpR"
/note="beta-lactamase"
rep_origin 14170..14758
/direction=RIGHT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"