Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V001425 | pXR002: EF1a-dCasRx-2A-EGFP | 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:
- pXR002: EF1a-dCasRx-2A-EGFP
- Antibiotic Resistance:
- Ampicillin
- Length:
- 12991 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Selection Marker:
- GFP
- Copy Number:
- High Copy
- Promoter:
- EF-1α
pXR002: EF1a-dCasRx-2A-EGFP vector 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.
pXR002: EF1a-dCasRx-2A-EGFP vector Sequence
LOCUS V001425 12991 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V001425
VERSION V001425
KEYWORDS pXR002: EF1a-dCasRx-2A-EGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 12991)
AUTHORS Konermann S, Lotfy P, Brideau NJ, Oki J, Shokhirev MN, Hsu PD
TITLE Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR
Effectors.
JOURNAL Cell. 2018 Mar 8. pii: S0092-8674(18)30207-1. doi:
10.1016/j.cell.2018.02.033.
PUBMED 29551272
REFERENCE 2 (bases 1 to 12991)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 12991)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2018
Mar 8. pii: S0092-8674(18)30207-1. doi: 10.1016/j.cell.2018.02.033."
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..12991
/mol_type="other DNA"
/organism="synthetic DNA construct"
polyA_signal complement(60..284)
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
LTR complement(316..496)
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
primer_bind 1001..1017
/label="pBluescriptKS"
/note="For pBluescript vector"
primer_bind 1002..1018
/label="KS primer"
/note="common sequencing primer, one of multiple similar
variants"
misc_feature complement(1021..1609)
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
CDS complement(1637..2350)
/label="EGFP"
/note="enhanced GFP"
CDS complement(2351..2404)
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS complement(2420..2446)
/label="HA"
/note="HA (human influenza hemagglutinin) epitope tag"
CDS complement(2456..2476)
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
CDS complement(2486..5383)
/label="RfxCas13d"
/note="RNA-targeting Type VI CRISPR protein from
Ruminococcus flavefaciens strain XPD3002 (Konermann et al.,
2018)"
CDS complement(5393..5413)
/label="SV40 NLS"
/note="nuclear localization signal of SV40 (simian virus
40) large T antigen"
promoter complement(5432..6610)
/label="EF-1-alpha promoter"
/note="strong constitutive promoter for human elongation
factor EF-1-alpha"
misc_feature complement(6735..6852)
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
CDS complement(6960..7001)
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
CDS complement(7150..7194)
/label="gp41 peptide"
/note="antigenic peptide corresponding to amino acids 655
to 669 of the HIV envelope protein gp41 (Lutje Hulsik et
al., 2013)"
misc_feature complement(7379..7612)
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature complement(8105..8230)
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
LTR complement(8277..8457)
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
promoter complement(8472..8674)
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
enhancer complement(8675..9054)
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
primer_bind 9226..9245
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"
promoter 9320..9424
/label="AmpR promoter"
CDS 9425..10282
/label="AmpR"
/note="beta-lactamase"
rep_origin 10456..11044
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 11198..11215
/label="L4440"
/note="L4440 vector, forward primer"
protein_bind 11332..11353
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 11368..11398
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 11406..11422
/label="lac operator"
/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)."
primer_bind 11430..11446
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
polyA_signal complement(11483..11616)
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
CDS complement(11749..12120)
/label="BleoR"
/note="antibiotic-binding protein"
promoter complement(12139..12186)
/label="EM7 promoter"
/note="synthetic bacterial promoter"
promoter complement(12234..12563)
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
primer_bind complement(12687..12708)
/label="F1ori-F"
/note="F1 origin, forward primer"
primer_bind 12899..12918
/label="F1ori-R"
/note="F1 origin, reverse primer"