LOCUS       sequence-392289-        7420 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Stable and dox. inducible expression of Trim69 isoform D (codon 
            optimized) after retroviral-mediated gene transfer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7420)
  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 7420)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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 D (codon opti...
FEATURES             Location/Qualifiers
     source          1..7420
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        83..462
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        463..666
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             667..842
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    905..1104
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1305..1721
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1651..1673
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1689..1705
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1757..2071
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1765..1783
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1801..1819
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1836..1854
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1872..1890
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1908..1926
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1943..1961
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1979..1997
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2044..2068
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2091..2114
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        2980..3479
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3015..3038)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3412..3431
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3500..4099
                     /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(3500..3519)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3996..4016
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4280..4705
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(4808..4830)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4966..4986)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4971..5300
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5151..5286
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5322..5341
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5422..5439)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5593..6181)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5673..5692)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6352..7143,7144..7212))
                     /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     6975..6994
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7213..7317)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7385..7403
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttagcttggc cattgcatac gttgtatcca tatcataata tgtacattta tattggctca
       61 tgtccaacat taccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt
      121 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
      181 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
      241 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
      301 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
      361 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
      421 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
      481 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
      541 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
      601 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
      661 agagctcaat aaaagagccc acaacccctc actcggcgcg ccagtcttcc gatagactgc
      721 gtcgcccggg tacccgtatt cccaataaag cctcttgctg tttgcatccg aatcgtggtc
      781 tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccacgacgg gggtctttca
      841 tttgggggct cgtccgggat ttggagaccc ctgcccaggg accaccgacc caccaccggg
      901 aggtaagctg gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgtttgatg
      961 ttatgcgcct gcgtctgtac tagttagcta actagctctg tatctggcgg acccgtggtg
     1021 gaactgacga gttctgaaca cccggccgca accctgggag acgtcccagg gactttgggg
     1081 gccgtttttg tggcccgacc tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat
     1141 atgtggttct ggtaggagac gagaacctaa aacagttccc gcctccgtct gaatttttgc
     1201 tttcggtttg gaaccgaagc cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt
     1261 gttgtctctg tctgactgtg tttctgtatt tgtctgaaaa ttagggccag actgttacca
     1321 ctcccttaag tttgacctta ggtcactgga aagatgtcga gcggatcgct cacaaccagt
     1381 cggtagatgt caagaagaga cgttgggtta ccttctgctc tgcagaatgg ccaaccttta
     1441 acgtcggatg gccgcgagac ggcaccttta accgagacct catcacccag gttaagatca
     1501 aggtcttttc acctggcccg catggacacc cagaccaggt cccctacatc gtgacctggg
     1561 aagccttggc ttttgacccc cctccctggg tcaagccctt tgtacaccct aagcctccgc
     1621 ctcctcttcc tccatccgcc ccgtctctcc cccttgaacc tcctcgttcg accccgcctc
     1681 gatcctccct ttatccagcc ctcactcctt ctctaggcgc cggaattgaa gatctgaggc
     1741 cctttcgtct tcactcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac
     1801 tccctatcag tgatagagaa cgatgtcgag tttactccct atcagtgata gagaacgtat
     1861 gtcgagttta ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga
     1921 tagagaacgt atgtcgagtt tatccctatc agtgatagag aacgtatgtc gagtttactc
     1981 cctatcagtg atagagaacg tatgtcgagg taggcgtgta cggtgggagg cctatataag
     2041 cagagctcgt ttagtgaacc gtcagatcgc ctggagaagg atccaccatg gactacaaag
     2101 acgatgacga taagggtggc ggagcggccg ccgagttcct caagctccat cagttcctgc
     2161 actccaagga aaaggatatc ctcaccgagc tgagagagga aggcaaagcc ctgaatgaag
     2221 agatggagct gaacctgagc cagctccagg agcagtgcct gctggccaag gacatgctcg
     2281 atattaccac cctgctgcac tccctggagc agggcatgaa ggtcctggcc accagagagc
     2341 tcatttccag gaagctgaac ctgggccagt ataagggccc cattcagtac atggtgtgga
     2401 gggagatgca ggacaccctg tgtcccggcc tgagccctct gacactggac cccaaaacag
     2461 cccaccccaa tctggtgctg tccaagagcc agacctccgt gtggcatggc gacatcaaga
     2521 agatcatgcc cgatgacccc gagaggtttg acagcagcgt cgctgtgctg ggctccagag
     2581 gctttaccag cggcaaatgg tactgggaag tggaggtggc caaaaagacc aaatggacag
     2641 tgggagtggt gagggaaagc attatcagga agggcagctg tcctctgaca cctgagcagg
     2701 gcttttggct gctgaggctg agaaaccaga cagacctgaa ggccctggac ctccccagct
     2761 tcagcctgac cctgacaaac aacctggaca aggtcggcat ctacctggac tacgagggag
     2821 gccagctgag cttctataat gccaaaacca tgacacacat ctataccttt tccaacacat
     2881 ttatggagaa gctgtaccct tatttctgtc cctgcctgaa cgacggcggc gaaaataagg
     2941 agcccctgca cattctgcac ccccagtgag aattctaccg ggtaggggag gcgcttttcc
     3001 caaggcagtc tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg
     3061 gcctctggcc tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg
     3121 gtggcccctt cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc
     3181 agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga
     3241 tggacagcac cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc
     3301 tttgctcctt cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca
     3361 ggggcgggct caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca
     3421 cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcga
     3481 cctgcagccc aagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga
     3541 cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg
     3601 ccacaccgtc gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct
     3661 cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc
     3721 ggtctggacc acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg
     3781 catggccgag ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc
     3841 gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca
     3901 ccagggcaag ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc
     3961 cggggtgccc gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct
     4021 cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac
     4081 ccgcaagccc ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc
     4141 acgaccccat gcatcggcgt ctcgagatat cagtggtcca ggctctagtt ttgactcaac
     4201 aatatcacca gctgaagcct atagagtacg agccatagat aaaataaaag attttattta
     4261 gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag
     4321 taacgccatt ttgcaaggca tggaaaaata cataactgag aatagagaag ttcagatcaa
     4381 ggtcaggaac agatggaaca gggtcgaccc tagagaacca tcagatgttt ccagggtgcc
     4441 ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct
     4501 tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggg
     4561 gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg
     4621 cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac
     4681 tacccgtcag cgggggtctt tcatttgggg gctcgtccgg gatcgggaga cccctgccca
     4741 gggaccaccg acccaccacc gggaggtaag ctggctgcct cgcgcgtttc ggtgatgacg
     4801 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     4861 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     4921 ccatgaccca gtcacgtagc gatagcggag tgtagatccg gctgtggaat gtgtgtcagt
     4981 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     5041 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     5101 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     5161 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     5221 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     5281 gaggcctagg cttttgcaaa aagcttactg gcttaactat gcggcatcag agcagattgt
     5341 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     5401 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     5461 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5521 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5581 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5641 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5701 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5761 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5821 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5881 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5941 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6001 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     6061 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6121 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6181 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6241 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6301 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     6361 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     6421 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     6481 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     6541 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     6601 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     6661 cattgctgca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     6721 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     6781 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     6841 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     6901 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     6961 ggcgtcaaca cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7021 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7081 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7141 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7201 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7261 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7321 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     7381 taaaaatagg cgtatcacga ggccctttcg tcttcaagaa
//
