Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V005497 | pZLCv2-3xFLAG-dCas9-HA-2xNLS | 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:
- pZLCv2-3xFLAG-dCas9-HA-2xNLS
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14962 bp
- Type:
- Mammalian Expression, Lentiviral, CRISPR
- Replication origin:
- ori
- Selection Marker:
- Puromycin
- Copy Number:
- High Copy
- Promoter:
- U6
- Cloning Method:
- Restriction Enzyme
pZLCv2-3xFLAG-dCas9-HA-2xNLS 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.
pZLCv2-3xFLAG-dCas9-HA-2xNLS vector Sequence
LOCUS V005497 14962 bp DNA circular SYN 13-MAY-2021
DEFINITION Exported.
ACCESSION V005497
VERSION V005497
KEYWORDS pZLCv2-3xFLAG-dCas9-HA-2xNLS
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14962)
AUTHORS Campbell AE, Shadle SC, Jagannathan S, Lim JW, Resnick R, Tawil R,
van der Maarel SM, Tapscott SJ
TITLE NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by
DUX4-induced MBD3L proteins.
JOURNAL Elife. 2018 Mar 13;7. pii: 31023. doi: 10.7554/eLife.31023.
PUBMED 29533181
REFERENCE 2 (bases 1 to 14962)
TITLE Direct Submission
REFERENCE 3 (bases 1 to 14962)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Elife.";
date: "2018-03-13"; pages: "
10.7554/eLife.31023"
SGRef: number: 2; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..14962
/mol_type="other DNA"
/organism="synthetic DNA construct"
enhancer 1..380
/label="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
promoter 381..583
/label="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 598..778
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 825..950
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1443..1676
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 1861..1905
/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 2054..2095
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 2203..2320
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2371..2611
/label="U6 promoter"
/note="RNA polymerase III promoter for human U6 snRNA"
misc_RNA 4501..4576
/label="gRNA scaffold"
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 4638..4849
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 4870..4935
/label="3xFLAG"
/note="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
CDS 4942..9042
/label="dCas9"
/note="catalytically dead mutant of the Cas9 endonuclease
from the Streptococcus pyogenes Type II CRISPR/Cas system"
CDS 9046..9072
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label="HA"
/translation="YPYDVPDYA"
CDS 9091..9111
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 9118..9138
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label="SV40 NLS"
/translation="PKKKRKV"
CDS 9148..9204
/label="P2A"
/note="2A peptide from porcine teschovirus-1 polyprotein"
CDS 9205..9798
/label="PuroR"
/note="puromycin N-acetyltransferase"
primer_bind complement(9870..9890)
/label="WPRE-R"
/note="WPRE, reverse primer"
CDS complement(10288..10299)
/label="Factor Xa site"
/note="Factor Xa recognition and cleavage site"
LTR 10477..10710
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 10742..10966
/label="bGH poly(A) signal"
/note="bovine growth hormone polyadenylation signal"
rep_origin 11012..11440
/label="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 11454..11783
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
promoter 11831..11878
/label="EM7 promoter"
/note="synthetic bacterial promoter"
CDS 11897..12268
/label="BleoR"
/note="antibiotic-binding protein"
polyA_signal 12401..12534
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
primer_bind complement(12571..12587)
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12571..12587)
/label="M13 Reverse"
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12584..12606)
/label="M13/pUC Reverse"
/note="In lacZ gene"
protein_bind 12595..12611
/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(12619..12649)
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind complement(12664..12685)
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12802..12819)
/label="L4440"
/note="L4440 vector, forward primer"
rep_origin complement(12973..13561)
/direction=LEFT
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13735..14592)
/label="AmpR"
/note="beta-lactamase"
promoter complement(14593..14697)
/label="AmpR promoter"
primer_bind complement(14772..14791)
/label="pRS-marker"
/note="pRS vectors, use to sequence yeast selectable
marker"