LOCUS       Exported               11634 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Tet-regulated (Tet-on) lentiviral vector for transgene (hPGK 
            promoter) - AND/OR - shRNA (H1 promoter when subcloned from pLVTHM 
            (Addgene#12247)) - 2nd generation.
ACCESSION   .
VERSION     .
KEYWORDS    pLVPT-tTR-KRAB
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11634)
  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 11634)
  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..11634
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_feature    294..411
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        450..949
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(485..508)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     882..901
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      1000..1009
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1006..1722
                     /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             1006..1722
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1051..1072)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1312..1331)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1659..1680
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1818..2369
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1962..1979)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2189..2208
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2372..2989
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MARLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG"
     primer_bind     complement(2445..2464)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             2993..3013
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3116..3310
                     /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(3449..3465)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3450..3466)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     misc_feature    3480..4068
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3533..3553)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3951..3962)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    4206..4476
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     LTR             4515..4695
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     promoter        complement(4720..4738)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(4721..4738)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5028..5047)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(5261..5280)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5380..5402
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5440..5458)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5526..5630
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5631..6491
                     /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(5849..5868)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6662..7250
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7151..7170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7404..7421
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(7491..7511)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7496..7825
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7676..7811
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     7738..7757
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     intron          8959..9024
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             9154..9174
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    9599..9733
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9636..9655)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9690..9709
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             9901..10534
                     /label=3 LTR
                     /note="3 long terminal repeat (LTR) from HIV-1"
     misc_feature    10581..10706
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    11199..11432
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             join(11617..11634,1..27)
                     /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"