LOCUS       Exported               13332 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  2nd generation lentiviral vector for reversible immortalization of 
            human primary cells.
ACCESSION   .
VERSION     .
KEYWORDS    pLOX-TERT-iresTK
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 13332)
  AUTHORS   Salmon P, Oberholzer J, Occhiodoro T, Morel P, Lou J, Trono D
  TITLE     Reversible immortalization of human primary cells by 
            lentivector-mediated transfer of specific genes.
  JOURNAL   Mol Ther. 2000 Oct . 2(4):404-14.
  PUBMED    11020357
REFERENCE   2  (bases 1 to 13332)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Mol Ther. 
            2000 Oct . 2(4):404-14."
FEATURES             Location/Qualifiers
     source          1..13332
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     LTR             92..725
                     /label=3 LTR
                     /note="3 long terminal repeat (LTR) from HIV-1"
     misc_feature    772..897
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1390..1623
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1808..1852
                     /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"
     enhancer        2131..2434
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2435..2638
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2589..2609
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2635..2659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2782..6180
                     /codon_start=1
                     /gene="TERT"
                     /product="human telomerase reverse transcriptase, the 
                     catalytic subunit of telomerase"
                     /label=hTERT
                     /note="prevents telomere shortening and allows 
                     postsenescent cells to proliferate beyond crisis (Counter 
                     et al., 1998); also known as EST2"
                     /translation="MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPA
                     AFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALL
                     DGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVA
                     PSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGAR
                     RRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEE
                     ATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQ
                     LRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGN
                     HAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSP
                     WQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRGC
                     AWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFY
                     RKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVN
                     MDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFV
                     LRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHG
                     HVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRF
                     MCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFL
                     LVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPW
                     CGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDL
                     QVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILK
                     AKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKL
                     PGTTLTALEAAANPALPSDFKTILD"
     misc_feature    6233..6694
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6289..6306)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     6516..6535
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             6699..7829
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQGGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRPMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     protein_bind    7985..8018
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     LTR             8038..8218
                     /label=5 LTR (truncated)
                     /note="truncated 5 long terminal repeat (LTR) from HIV-1"
     promoter        complement(8243..8261)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(8244..8261)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(8551..8570)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(8784..8803)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8903..8925
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8963..8981)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        9049..9153
                     /gene="bla"
                     /label=AmpR promoter
     CDS             9154..10014
                     /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(9372..9391)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      10185..10773
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10674..10693
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     10927..10944
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(11014..11034)
                     /label=pBABE 3
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        11019..11348
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      11199..11334
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     11261..11280
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     intron          12482..12547
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     CDS             12677..12697
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    13122..13256
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(13159..13178)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     13213..13232
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"