Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V000970 | lenti-EF1a-dCas9-VPR-Puro | 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:
- lenti-EF1a-dCas9-VPR-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 15702 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Selection Marker:
- Puromycin, Zeocin
- Copy Number:
- High Copy
- Promoter:
- EF-1α
- Cloning Method:
- Gibson Cloning
- 5' Primer:
- GTTTGGATCTTGGTTCATTCTCAAGCCTCAG
- 3' Primer:
- cacatagcgtaaaaggagcaacatag
lenti-EF1a-dCas9-VPR-Puro 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.
lenti-EF1a-dCas9-VPR-Puro vector Sequence
LOCUS V000970 15702 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V000970
VERSION V000970
KEYWORDS lenti-EF1a-dCas9-VPR-Puro
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 15702)
AUTHORS Ho SM, Hartley BJ, Flaherty E, Rajarajan P, Abdelaal R, Obiorah I,
Barretto N, Muhammad H, Phatnani HP, Akbarian S, Brennand KJ
TITLE Evaluating Synthetic Activation and Repression of
Neuropsychiatric-Related Genes in hiPSC-Derived NPCs, Neurons, and
Astrocytes.
JOURNAL Stem Cell Reports. 2017 Aug 8;9(2):615-628. doi:
10.1016/j.stemcr.2017.06.012. Epub 2017 Jul 27.
PUBMED 28757163
REFERENCE 2 (bases 1 to 15702)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 15702)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.stemcr.2017.06.012"; journalName: "Stem Cell Reports";
date: "2017-08-8- 8"; volume: "9"; issue: "2"; pages: "615-628"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..15702
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 130..1308
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
CDS 1339..5442
/label="Cas9m4"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 5455..5475
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
protein_bind 5512..5536
/gene="mutant version of attB"
/label="attB1"
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 5566..5715
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label="VP64"
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 5740..5760
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 6190..6546
/codon_start=1
/product="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
/label="RelA (p65) AD"
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALL"
CDS 6565..7134
/codon_start=1
/product="transcriptional activation domain from the human
herpesvirus 4 (Epstein-Barr virus) replication and
transcription activator Rta/BRLF1 (Hardwick et al., 1992;
Chavez et al., 2015)"
/label="Rta AD"
/translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
DTFLNDECLLHAMHISTGLSIFDTSLF"
CDS 7150..7206
/label="P2A"
/note="2A peptide from porcine teschovirus-1 polyprotein"
CDS 7219..7809
/label="PuroR"
/note="puromycin N-acetyltransferase"
misc_feature 7837..8425
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8428..8444)
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(8429..8445)
/label="pBluescriptKS"
/note="For pBluescript vector"
LTR 8950..9130
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
polyA_signal 9162..9386
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 9432..9860
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(9869..9889)
/label="pBABE 3'"
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 10007..10203
/label="SV40 promoter"
/note="SV40 early promoter"
promoter 10251..10298
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 10317..10688
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 10821..10954
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(10991..11007)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(10991..11007)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11004..11026)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 11015..11031
/label="lac operator"
/bound_moiety="lac repressor encoded by lacI"
/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)."
promoter complement(11039..11069)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(11084..11105)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11222..11239)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(11393..11981)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(12155..13012)
/label="AmpR"
/note="beta-lactamase"
promoter complement(13013..13117)
/label="AmpR promoter"
primer_bind complement(13192..13211)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 13383..13762
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 13763..13965
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 13980..14160
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 14207..14332
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 14825..15058
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 15243..15287
/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 15436..15477
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 15585..15702
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"