LOCUS       sequence-392223-        7606 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Stable and dox. inducible expression of Trim69 isoform B (codon 
            optimized) after retroviral-mediated gene transfer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7606)
  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 7606)
  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 B (codon opti...
FEATURES             Location/Qualifiers
     source          1..7606
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        91..470
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        471..674
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             675..850
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    913..1112
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1313..1729
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1659..1681
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1697..1713
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1765..2079
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1773..1791
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1809..1827
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1844..1862
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1880..1898
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1916..1934
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1951..1969
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1987..2005
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2052..2076
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2099..2122
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        3174..3673
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3209..3232)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3606..3625
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             3694..4293
                     /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(3694..3713)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4190..4210
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4474..4899
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5002..5024)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5160..5180)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5165..5494
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5345..5480
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5516..5535
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5616..5633)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5787..6375)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5867..5886)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6546..7337,7338..7406))
                     /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     7169..7188
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7407..7511)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7579..7597
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttcaagaatt agcttggcca ttgcatacgt tgtatccata tcataatatg tacatttata
       61 ttggctcatg tccaacatta ccgccatgtt gacattgatt attgactagt tattaatagt
      121 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      181 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      241 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      301 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      361 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      421 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      481 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      541 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      601 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      661 atataagcag agctcaataa aagagcccac aacccctcac tcggcgcgcc agtcttccga
      721 tagactgcgt cgcccgggta cccgtattcc caataaagcc tcttgctgtt tgcatccgaa
      781 tcgtggtctc gctgttcctt gggagggtct cctctgagtg attgactacc cacgacgggg
      841 gtctttcatt tgggggctcg tccgggattt ggagacccct gcccagggac caccgaccca
      901 ccaccgggag gtaagctggc cagcaactta tctgtgtctg tccgattgtc tagtgtctat
      961 gtttgatgtt atgcgcctgc gtctgtacta gttagctaac tagctctgta tctggcggac
     1021 ccgtggtgga actgacgagt tctgaacacc cggccgcaac cctgggagac gtcccaggga
     1081 ctttgggggc cgtttttgtg gcccgacctg aggaagggag tcgatgtgga atccgacccc
     1141 gtcaggatat gtggttctgg taggagacga gaacctaaaa cagttcccgc ctccgtctga
     1201 atttttgctt tcggtttgga accgaagccg cgcgtcttgt ctgctgcagc gctgcagcat
     1261 cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaatt agggccagac
     1321 tgttaccact cccttaagtt tgaccttagg tcactggaaa gatgtcgagc ggatcgctca
     1381 caaccagtcg gtagatgtca agaagagacg ttgggttacc ttctgctctg cagaatggcc
     1441 aacctttaac gtcggatggc cgcgagacgg cacctttaac cgagacctca tcacccaggt
     1501 taagatcaag gtcttttcac ctggcccgca tggacaccca gaccaggtcc cctacatcgt
     1561 gacctgggaa gccttggctt ttgacccccc tccctgggtc aagccctttg tacaccctaa
     1621 gcctccgcct cctcttcctc catccgcccc gtctctcccc cttgaacctc ctcgttcgac
     1681 cccgcctcga tcctcccttt atccagccct cactccttct ctaggcgccg gaattgaaga
     1741 tctgaggccc tttcgtcttc actcgagttt actccctatc agtgatagag aacgtatgtc
     1801 gagtttactc cctatcagtg atagagaacg atgtcgagtt tactccctat cagtgataga
     1861 gaacgtatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct
     1921 atcagtgata gagaacgtat gtcgagttta tccctatcag tgatagagaa cgtatgtcga
     1981 gtttactccc tatcagtgat agagaacgta tgtcgaggta ggcgtgtacg gtgggaggcc
     2041 tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagaaggat ccaccatgga
     2101 ctacaaagac gatgacgata agggtggcgg agcggccgcc gaggaggagc tggccattca
     2161 gcagggccag ctggagacca ccctgaagga gctgcaaacc ctcaggaaca tgcagaagga
     2221 ggccatcgcc gctcacaagg aaaacaagct gcacctccag cagcacgtgt ccatggagtt
     2281 cctcaagctc catcagttcc tgcactccaa ggaaaaggat atcctcaccg agctgagaga
     2341 ggaaggcaaa gccctgaatg aagagatgga gctgaacctg agccagctcc aggagcagtg
     2401 cctgctggcc aaggacatgc tcgtgagcat ccaggccaag accgaacagc agaacagctt
     2461 cgacttcctc aaggatatta ccaccctgct gcactccctg gagcagggca tgaaggtcct
     2521 ggccaccaga gagctcattt ccaggaagct gaacctgggc cagtataagg gccccattca
     2581 gtacatggtg tggagggaga tgcaggacac cctgtgtccc ggcctgagcc ctctgacact
     2641 ggaccccaaa acagcccacc ccaatctggt gctgtccaag agccagacct ccgtgtggca
     2701 tggcgacatc aagaagatca tgcccgatga ccccgagagg tttgacagca gcgtcgctgt
     2761 gctgggctcc agaggcttta ccagcggcaa atggtactgg gaagtggagg tggccaaaaa
     2821 gaccaaatgg acagtgggag tggtgaggga aagcattatc aggaagggca gctgtcctct
     2881 gacacctgag cagggctttt ggctgctgag gctgagaaac cagacagacc tgaaggccct
     2941 ggacctcccc agcttcagcc tgaccctgac aaacaacctg gacaaggtcg gcatctacct
     3001 ggactacgag ggaggccagc tgagcttcta taatgccaaa accatgacac acatctatac
     3061 cttttccaac acatttatgg agaagctgta cccttatttc tgtccctgcc tgaacgacgg
     3121 cggcgaaaat aaggagcccc tgcacattct gcacccccag tgagaattct accgggtagg
     3181 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     3241 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     3301 ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     3361 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     3421 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     3481 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     3541 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     3601 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     3661 tccgggcctt tcgacctgca gcccaagctt accatgaccg agtacaagcc cacggtgcgc
     3721 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     3781 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     3841 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     3901 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     3961 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     4021 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     4081 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     4141 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     4201 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     4261 acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg
     4321 accgaaagga gcgcacgacc ccatgcatcg gcgtctcgag atatcagtgg tccaggctct
     4381 agttttgact caacaatatc accagctgaa gcctatagag tacgagccat agataaaata
     4441 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     4501 caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga
     4561 gaagttcaga tcaaggtcag gaacagatgg aacagggtcg accctagaga accatcagat
     4621 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca
     4681 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca
     4741 acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc
     4801 aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc
     4861 tctgagtgat tgactacccg tcagcggggg tctttcattt gggggctcgt ccgggatcgg
     4921 gagacccctg cccagggacc accgacccac caccgggagg taagctggct gcctcgcgcg
     4981 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     5041 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     5101 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtaga tccggctgtg
     5161 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     5221 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     5281 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     5341 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     5401 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     5461 aggaggcttt tttggaggcc taggcttttg caaaaagctt actggcttaa ctatgcggca
     5521 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     5581 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     5641 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     5701 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     5761 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5821 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5881 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5941 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6001 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6061 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6121 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6181 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
     6241 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     6301 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6361 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6421 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6481 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     6541 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     6601 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     6661 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     6721 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6781 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6841 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6901 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6961 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     7021 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     7081 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     7141 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa
     7201 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     7261 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     7321 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     7381 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     7441 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     7501 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     7561 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
