Basic Vector Information
- Vector Name:
- pLV3-U6-MCS-sgRNA-EF1a-RfxCas13d-Puro
- Antibiotic Resistance:
- Ampicillin
- Length:
- 13637 bp
- Type:
- Gene knockout
- Replication origin:
- ori
- Host:
- Mammalian cells, Lentivirus
- Selection Marker:
- Puro
- Promoter:
- U6
- Growth Strain(s):
- Stbl3
- Growth Temperature:
- 37℃
pLV3-U6-MCS-sgRNA-EF1a-RfxCas13d-Puro 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 ℃.
pLV3-U6-MCS-sgRNA-EF1a-RfxCas13d-Puro vector Sequence
LOCUS 62056_15670 13637 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 13637) AUTHORS . TITLE Direct Submission FEATURES Location/Qualifiers source 1..13637 /mol_type="other DNA" /organism="synthetic DNA construct" enhancer 238..617 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 618..820 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" LTR 835..1015 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 1062..1187 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1680..1913 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 2098..2142 /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 2291..2332 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" misc_feature 2440..2557 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 2608..2848 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" misc_RNA 4738..4813 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 4875..5086 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" CDS 5111..8008 /label=RfxCas13d /note="RNA-targeting Type VI CRISPR protein from Ruminococcus flavefaciens strain XPD3002 (Konermann et al., 2018)" CDS 8015..8041 /label=NES /note="nuclear export signal from the HIV Rev protein (Fischer et al., 1995)" CDS 8060..8116 /label=P2A /note="2A peptide from porcine teschovirus-1 polyprotein" CDS 8117..8710 /label=PuroR /note="puromycin N-acetyltransferase" misc_feature 8729..9317 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" LTR 9389..9622 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 9654..9878 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 10366..10695 /label=SV40 promoter /note="SV40 enhancer and early promoter" promoter 10743..10790 /label=EM7 promoter /note="synthetic bacterial promoter" CDS 10809..11180 /label=BleoR /note="antibiotic-binding protein" polyA_signal 11313..11446 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" promoter complement(11531..11561) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind complement(11576..11597) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." rep_origin complement(11885..12473) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" CDS complement(12647..13504) /label=AmpR /note="beta-lactamase" promoter complement(13505..13609) /label=AmpR promoter
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