LOCUS       sequence-392270-        7471 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable and dox. inducible expression of Trim69 isoform C (codon 
            optimized) after retroviral-mediated gene transfer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7471)
  AUTHORS   Song Y, Nguyen XN, Kumar A, da Silva C, Picard L, Etienne L, 
            Cimarelli A
  TITLE     Trim69 is a microtubule regulator that acts as a pantropic viral 
            inhibitor.
  JOURNAL   Proc Natl Acad Sci U S A. 2022 Oct 25;119(43):e2211467119. doi: 
            10.1073/pnas.2211467119. Epub 2022 Oct 17.
  PUBMED    36251989
REFERENCE   2  (bases 1 to 7471)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2022-10-25"; volume: "119(43):e2211467119. 
            doi"; pages: " 10.1073/pnas.2211467119. Epub 2022 Oct 17"
COMMENT     Sequence Label: pRetroX-Tight-Pur-Trim69 isoform C (codon opti...
FEATURES             Location/Qualifiers
     source          1..7471
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        30..409
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        410..613
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             614..789
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    852..1051
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1252..1668
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1598..1620
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1636..1652
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1704..2018
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1712..1730
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1748..1766
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1783..1801
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1819..1837
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1855..1873
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1890..1908
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1926..1944
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1991..2015
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2038..2061
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        2978..3477
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3013..3036)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3410..3429
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3498..4097
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3498..3517)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3994..4014
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4278..4703
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(4806..4828)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4964..4984)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4969..5298
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5149..5284
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5320..5339
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5420..5437)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5591..6179)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5671..5690)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6350..7141,7142..7210))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6973..6992
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7211..7315)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7383..7401
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tggctcatgt ccaacattac cgccatgttg acattgatta ttgactagtt attaatagta
       61 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      121 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      181 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      241 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      301 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      361 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
      421 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
      481 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
      541 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
      601 tataagcaga gctcaataaa agagcccaca acccctcact cggcgcgcca gtcttccgat
      661 agactgcgtc gcccgggtac ccgtattccc aataaagcct cttgctgttt gcatccgaat
      721 cgtggtctcg ctgttccttg ggagggtctc ctctgagtga ttgactaccc acgacggggg
      781 tctttcattt gggggctcgt ccgggatttg gagacccctg cccagggacc accgacccac
      841 caccgggagg taagctggcc agcaacttat ctgtgtctgt ccgattgtct agtgtctatg
      901 tttgatgtta tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc
      961 cgtggtggaa ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac
     1021 tttgggggcc gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg
     1081 tcaggatatg tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa
     1141 tttttgcttt cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc
     1201 gttctgtgtt gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact
     1261 gttaccactc ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac
     1321 aaccagtcgg tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca
     1381 acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt
     1441 aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg
     1501 acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag
     1561 cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc
     1621 ccgcctcgat cctcccttta tccagccctc actccttctc taggcgccgg aattgaagat
     1681 ctgaggccct ttcgtcttca ctcgagttta ctccctatca gtgatagaga acgtatgtcg
     1741 agtttactcc ctatcagtga tagagaacga tgtcgagttt actccctatc agtgatagag
     1801 aacgtatgtc gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta
     1861 tcagtgatag agaacgtatg tcgagtttat ccctatcagt gatagagaac gtatgtcgag
     1921 tttactccct atcagtgata gagaacgtat gtcgaggtag gcgtgtacgg tgggaggcct
     1981 atataagcag agctcgttta gtgaaccgtc agatcgcctg gagaaggatc caccatggac
     2041 tacaaagacg atgacgataa gggtggcgga gcggccgccg agttcctcaa gctccatcag
     2101 ttcctgcact ccaaggaaaa ggatatcctc accgagctga gagaggaagg caaagccctg
     2161 aatgaagaga tggagctgaa cctgagccag ctccaggagc agtgcctgct ggccaaggac
     2221 atgctcgtga gcatccaggc caagaccgaa cagcagaaca gcttcgactt cctcaaggat
     2281 attaccaccc tgctgcactc cctggagcag ggcatgaagg tcctggccac cagagagctc
     2341 atttccagga agctgaacct gggccagtat aagggcccca ttcagtacat ggtgtggagg
     2401 gagatgcagg acaccctgtg tcccggcctg agccctctga cactggaccc caaaacagcc
     2461 caccccaatc tggtgctgtc caagagccag acctccgtgt ggcatggcga catcaagaag
     2521 atcatgcccg atgaccccga gaggtttgac agcagcgtcg ctgtgctggg ctccagaggc
     2581 tttaccagcg gcaaatggta ctgggaagtg gaggtggcca aaaagaccaa atggacagtg
     2641 ggagtggtga gggaaagcat tatcaggaag ggcagctgtc ctctgacacc tgagcagggc
     2701 ttttggctgc tgaggctgag aaaccagaca gacctgaagg ccctggacct ccccagcttc
     2761 agcctgaccc tgacaaacaa cctggacaag gtcggcatct acctggacta cgagggaggc
     2821 cagctgagct tctataatgc caaaaccatg acacacatct ataccttttc caacacattt
     2881 atggagaagc tgtaccctta tttctgtccc tgcctgaacg acggcggcga aaataaggag
     2941 cccctgcaca ttctgcaccc ccagtgagaa ttctaccggg taggggaggc gcttttccca
     3001 aggcagtctg gagcatgcgc tttagcagcc ccgctgggca cttggcgcta cacaagtggc
     3061 ctctggcctc gcacacattc cacatccacc ggtaggcgcc aaccggctcc gttctttggt
     3121 ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc cgccccgcag
     3181 ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct cgtgcagatg
     3241 gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca atagcagctt
     3301 tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg cgggctcagg
     3361 ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc attctgcacg
     3421 cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg cctttcgacc
     3481 tgcagcccaa gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     3541 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     3601 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     3661 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     3721 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     3781 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     3841 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     3901 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     3961 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     4021 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     4081 gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac
     4141 gaccccatgc atcggcgtct cgagatatca gtggtccagg ctctagtttt gactcaacaa
     4201 tatcaccagc tgaagcctat agagtacgag ccatagataa aataaaagat tttatttagt
     4261 ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta
     4321 acgccatttt gcaaggcatg gaaaaataca taactgagaa tagagaagtt cagatcaagg
     4381 tcaggaacag atggaacagg gtcgacccta gagaaccatc agatgtttcc agggtgcccc
     4441 aaggacctga aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc
     4501 tgttcgcgcg cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggggc
     4561 gccagtcctc cgattgactg agtcgcccgg gtacccgtgt atccaataaa ccctcttgca
     4621 gttgcatccg acttgtggtc tcgctgttcc ttgggagggt ctcctctgag tgattgacta
     4681 cccgtcagcg ggggtctttc atttgggggc tcgtccggga tcgggagacc cctgcccagg
     4741 gaccaccgac ccaccaccgg gaggtaagct ggctgcctcg cgcgtttcgg tgatgacggt
     4801 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     4861 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     4921 atgacccagt cacgtagcga tagcggagtg tagatccggc tgtggaatgt gtgtcagtta
     4981 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     5041 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     5101 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     5161 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     5221 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     5281 ggcctaggct tttgcaaaaa gcttactggc ttaactatgc ggcatcagag cagattgtac
     5341 tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca
     5401 tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5461 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5521 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5581 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5641 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5701 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5761 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5821 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5881 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5941 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6001 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     6061 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6121 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6181 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6241 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6301 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6361 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6421 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6481 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6541 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6601 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6661 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6721 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     6781 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     6841 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     6901 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     6961 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7021 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7081 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7141 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7201 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7261 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7321 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata
     7381 aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt agcttggcca ttgcatacgt
     7441 tgtatccata tcataatatg tacatttata t
//
