Basic Vector Information
3rd generation lentiviral plasmid for inducible expression of shRNA; puromycin selection.
Tet-pLKO-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 ℃.
Tet-pLKO-puro vector Sequence
LOCUS Tet-pLKO-puro. 10633 bp DNA circular SYN 11-SEP-2021 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS Tet-pLKO-puro. SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10633) TITLE Direct Submission REFERENCE 2 (bases 1 to 10633) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..10633 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 7..507 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter" intron 516..1088 /label=beta-globin intron /note="intron from rabbit beta-globin gene" promoter 1143..1161 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" CDS 1172..1792 /label=TetR /note="tetracycline repressor TetR" misc_feature 1827..2400 /label=IRES /note="internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV)" CDS 2420..3016 /label=PuroR /note="puromycin N-acetyltransferase" LTR 3147..3380 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 3452..3586 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" rep_origin 3613..3748 /label=SV40 ori /note="SV40 origin of replication" promoter complement(3769..3787) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3797..3813) /label=M13 fwd /note="M13 fwd" /note="common sequencing primer, one of multiple similar variants" rep_origin 3955..4410 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 4436..4540 /label=AmpR promoter CDS 4541..5398 /label=AmpR /note="beta-lactamase" rep_origin 5572..6160 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind 6448..6469 /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." promoter 6484..6514 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 6522..6538 /label=lac repressor encoded by lacI binding site /bound_moiety="lac repressor encoded by lacI" /note="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 6546..6562 /label=M13 rev /note="M13 rev" /note="common sequencing primer, one of multiple similar variants" promoter 6583..6601 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 6629..6855 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 6856..7036 /label=5' LTR (truncated) /note="5' LTR (truncated)" /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 7083..7208 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 7701..7934 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 8119..8163 /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 8312..8353 /note="Protein Tat from Human immunodeficiency virus type 1 group M subtype B (isolate WMJ22). Accession#: P12509" promoter 8452..8546 /label=H1-2O2 promoter /note="doxycycline-inducible variant of the human H1 RNA promoter (Henriksen et al., 2007)" promoter 10000..10507 /label=hPGK promoter /note="human phosphoglycerate kinase 1 promoter"
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