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 Resuspension 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 ℃.
EF1a-Cas9-2XNES-2ERT2 vector Sequence
LOCUS 62056_656 14418 bp DNA circular SYN 01-JAN-1980 DEFINITION synthetic circular DNA. ACCESSION . VERSION . 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" misc_feature 14278..14395 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1"
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