Price Information
| Cat No. | Plasmid Name | Availability | Add to cart |
|---|---|---|---|
| V017081 | EF1a-Cas9-2XNES-2ERT2 | 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:
- EF1a-Cas9-2XNES-2ERT2
- Antibiotic Resistance:
- Ampicillin
- Length:
- 14418 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Zeo
- Promoter:
- SV40
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
EF1a-Cas9-2XNES-2ERT2 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.
EF1a-Cas9-2XNES-2ERT2 vector Sequence
LOCUS V017081 14418 bp DNA circular SYN 01-JAN-1980
DEFINITION Exported.
ACCESSION V017081
VERSION V017081
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
.
REFERENCE 1 (bases 1 to 14418)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..14418
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 130..341
/label="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
CDS 1226..5326
/label="Cas9"
/note="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
CDS 5465..6400
/label="ERT2"
/note="mutated ligand-binding domain of the human estrogen
receptor (Feil et al., 1997)"
promoter 7542..7871
/label="SV40 promoter"
/note="SV40 enhancer and early promoter"
CDS 7881..8252
/label="BleoR"
/note="antibiotic-binding protein"
misc_feature 8341..8929
/label="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 9016..9249
/label="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 9321..9455
/label="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
promoter complement(9617..9635)
/label="T7 promoter"
/note="promoter for bacteriophage T7 RNA polymerase"
promoter 10284..10388
/label="AmpR promoter"
CDS 10389..11246
/label="AmpR"
/note="beta-lactamase"
rep_origin 11420..12008
/label="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind 12296..12317
/label="CAP binding site"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 12332..12362
/label="lac promoter"
/note="promoter for the E. coli lac operon"
protein_bind 12370..12386
/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 12394..12410
/label="M13 rev"
/note="common sequencing primer, one of multiple similar
variants"
promoter 12431..12449
/label="T3 promoter"
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 12477..12703
/label="RSV promoter"
/note="Rous sarcoma virus enhancer/promoter"
LTR 12704..12884
/label="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 12931..13056
/label="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 13549..13782
/label="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 13967..14011
/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 14160..14201
/note="Protein Tat from Human immunodeficiency virus type 1
group M subtype B (isolate WMJ22). Accession#: P12509"
/label="Protein Tat"
misc_feature 14278..14395
/label="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"