Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V012635 | pHR-SFFV-dCas9-BFP-KRAB | 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
Human expression vector containing SFFV promoter, dCas9 that is fused to 2x NLS, tagBFP and a KRAB domain
- Vector Name:
- pHR-SFFV-dCas9-BFP-KRAB
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14167 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Copy Number:
- High Copy
- Promoter:
- SFFV
- Cloning Method:
- Ligation Independent Cloning
- 5' Primer:
- gcttcccgagctctataaaagag
- 3' Primer:
- CCAGAGGTTGATTATCGATAAGC
pHR-SFFV-dCas9-BFP-KRAB 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.
pHR-SFFV-dCas9-BFP-KRAB vector Sequence
LOCUS V012635 14167 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V012635
VERSION V012635
KEYWORDS pHR-SFFV-dCas9-BFP-KRAB
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14167)
AUTHORS Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE,
Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA,
Weissman JS, Qi LS
TITLE CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in
Eukaryotes.
JOURNAL Cell. 2013 Jul 9. pii: S0092-8674(13)00826-X. doi:
10.1016/j.cell.2013.06.044.
PUBMED 23849981
REFERENCE 2 (bases 1 to 14167)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14167)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2013
Jul 9. pii: S0092-8674(13)00826-X. doi: 10.1016/j.cell.2013.06.044."
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14167
/mol_type="other DNA"
/organism="synthetic DNA construct"
LTR 364..621
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 668..793
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1290..1523
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1708..1752
/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 1901..1942
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2037..2154
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2307..2714
/label="SFFV promoter"
/note="spleen focus-forming virus long terminal repeat
(LTR) promoter"
CDS 2789..6892
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 6896..6922
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label="HA"
/translation="YPYDVPDYA"
CDS 6941..6961
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 6968..6988
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 7028..7723
/label="TagBFP"
/note="monomeric blue fluorescent protein"
CDS 7769..7954
/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"
misc_feature 8018..8606
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8609..8625)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8610..8626)
/label="pBluescriptKS"
/note="For pBluescript vector"
LTR 8716..8949
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
promoter complement(8974..8992)
/label="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind complement(9282..9301)
/label="pBRrevBam"
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
primer_bind complement(9515..9534)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 9634..9656
/label="pGEX 3'"
/note="pGEX vectors, reverse primer"
primer_bind complement(9694..9712)
/label="pBRforEco"
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 9780..9884
/label="AmpR promoter"
LTR 10000..10621
/label="3' LTR"
/note="3' long terminal repeat (LTR) from HIV-1"
rep_origin 10916..11504
/direction=RIGHT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 11658..11675
/label="L4440"
/note="L4440 vector, forward primer"
promoter 11750..12079
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
intron 13213..13278
/label="small t intron"
/note="SV40 (simian virus 40) small t antigen intron"
CDS 13408..13428
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
polyA_signal 13853..13987
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"