LOCUS       Exported               11645 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Tet-regulated (Tet-off) lentiviral vector for transgene (hPGK 
            promoter) - AND/OR - shRNA (H1 promoter when subcloned from pLVTHM 
            (Addgene#12247)) - 2nd generation.
ACCESSION   .
VERSION     .
KEYWORDS    pLVPT-rtTR-KRAB-2SM2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11645)
  AUTHORS   Szulc J, Wiznerowicz M, Sauvain MO, Trono D, Aebischer P
  TITLE     A versatile tool for conditional gene expression and knockdown.
  JOURNAL   Nat Methods. 2006 Feb . 3(2):109-16.
  PUBMED    16432520
REFERENCE   2  (bases 1 to 11645)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2006 Feb . 3(2):109-16."
FEATURES             Location/Qualifiers
     source          1..11645
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             12..645
                     /label=3 LTR
                     /note="3 long terminal repeat (LTR) from HIV-1"
     misc_feature    692..817
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1310..1543
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1727..1771
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    2038..2155
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2194..2693
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2229..2252)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2626..2645
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2744..2753
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2750..3466
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             2750..3466
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2795..2816)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3056..3075)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3403..3424
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3562..4113
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3706..3723)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3933..3952
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4737..4757
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4860..5054
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     primer_bind     complement(5192..5208)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5193..5209)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     misc_feature    5235..5823
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5288..5308)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5706..5717)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    5961..6231
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     LTR             6270..6450
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     promoter        complement(6475..6493)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(6476..6493)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(6783..6802)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(7016..7035)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7135..7157
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(7195..7213)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7281..7385
                     /gene="bla"
                     /label=AmpR promoter
     CDS             7386..8246
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(7604..7623)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8417..9005
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8906..8925
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     9159..9176
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(9246..9266)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        9251..9580
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      9431..9566
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     9493..9512
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     intron          10714..10779
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             10909..10929
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    11354..11488
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(11391..11410)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     11445..11464
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"