LOCUS       sequence-366483-        8652 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doxycycline-dependent expression of catalytically inactive NS3/4A 
            protease gene (S140A) in mammalian cells by retroviral transduction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8652)
  AUTHORS   Mosallanejad K, Kennedy SN, Bahleda KM, Slavik KM, Zhou W, Govande 
            AA, Hancks DC, Kranzusch PJ, Kagan JC
  TITLE     Species-specific self-DNA detection mechanisms by mammalian cyclic 
            GMP-AMP synthases.
  JOURNAL   Sci Immunol. 2023 Jan 20;8(79):eabp9765. doi: 
            10.1126/sciimmunol.abp9765. Epub 2023 Jan 20.
  PUBMED    36662885
REFERENCE   2  (bases 1 to 8652)
  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: "Sci 
            Immunol."; date: "2023-01-20"; volume: "8(79):eabp9765. doi"; pages:
            " 10.1126/sciimmunol.abp9765. Epub 2023 Jan 20"
COMMENT     Sequence Label: pTRE3G-NS3/4A S139A
FEATURES             Location/Qualifiers
     source          1..8652
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        330..533
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             534..709
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    772..971
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1172..1588
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1518..1540
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1556..1572
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1628..1977
                     /label=TRE3GV promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, optimized for 
                     retroviral and lentiviral vectors"
     protein_bind    1636..1654
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1672..1690
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1708..1726
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1744..1762
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1780..1798
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1816..1834
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1852..1870
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1916..1940
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1984..1993
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        4081..4578
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4116..4139)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4511..4530
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4599..5198
                     /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(4599..4618)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5095..5115
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5379..5804
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5907..5929)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6065..6085)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6070..6399
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6250..6385
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6421..6440
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6521..6538)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6692..7280)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6772..6791)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7451..8242,8243..8311))
                     /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     8074..8093
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8312..8416)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8484..8502
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(8602..8652,1..329)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
       61 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      121 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      181 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      241 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      301 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      361 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      421 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      481 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctcaataaa
      541 agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc gcccgggtac
      601 ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg ctgttccttg
      661 ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt gggggctcgt
      721 ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg taagctggcc
      781 agcaacttat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg
      841 tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt
      901 ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg
      961 cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt
     1021 aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa
     1081 ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct
     1141 gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt
     1201 gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa
     1261 gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc
     1321 gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc
     1381 tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt
     1441 tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc
     1501 atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta
     1561 tccagccctc actccttctc taggcgccgg aattgaagat ctcgatgagg ccctttcgtc
     1621 ttcactcgag tttactccct atcagtgata gagaacgtat gaagagttta ctccctatca
     1681 gtgatagaga acgtatgcag actttactcc ctatcagtga tagagaacgt ataaggagtt
     1741 tactccctat cagtgataga gaacgtatga ccagtttact ccctatcagt gatagagaac
     1801 gtatctacag tttactccct atcagtgata gagaacgtat atccagttta ctccctatca
     1861 gtgatagaga acgtatgtcg aggtaggcgt gtacggtggg cgcctataaa agcagagctc
     1921 gtttagtgaa ccgtcagatc gcctggagca attccacaac acttttgtct tatacttgga
     1981 tccgccacca tggcgcctat cacggcctac tcccaacaga cgcgaggcct acttggctgc
     2041 atcatcacta gcctcacagg ccgggacagg aaccaggtcg agggggaggt ccaagtggtc
     2101 tccaccgcaa cacaatcttt cctggcgacc tgcgtcaatg gcgtgtgttg gactgtctat
     2161 catggtgccg gctcaaagac ccttgccggc ccaaagggcc caatcaccca aatgtacacc
     2221 aatgtggacc aggacctcgt cggctggcaa gcgccccccg gggcgcgttc cttgacacca
     2281 tgcacctgcg gcagctcgga cctttacttg gtcacgaggc atgccgatgt cattccggtg
     2341 cgccggcggg gcgacagcag ggggagccta ctctccccca ggcccgtctc ctacttgaag
     2401 ggctccgcgg gcggtccact gctctgcccc tcggggcacg ctgtgggcat ctttcgggct
     2461 gccgtgtgca cccgaggggt tgcgaaggcg gtggactttg tacccgtcga gtctatggga
     2521 accactatgc ggtccccggt cttcacggac aactcgtccc ctccggccgt accgcagaca
     2581 ttccaggtgg cccatctaca cgcccctact ggtagcggca agagcactaa ggtgccggct
     2641 gcgtatgcag cccaagggta taaggtgctt gtcctgaacc cgtccgtcgc cgccacccta
     2701 ggtttcgggg cgtatatgtc taaggcacat ggtatcgacc ctaacatcag aatcggggta
     2761 aggaccatca ccacgggtgc ccccatcacg tactccacct atggcaagtt tcttgccgac
     2821 ggtggttgct ctgggggcgc ctatgacatc ataatatgtg atgagtgcca ctcaactgac
     2881 tcgaccacta tcctgggcat cggcacagtc ctggaccaag cggagacggc tggagcgcga
     2941 ctcgtcgtgc tcgccaccgc tacgcctccg ggatcggtca ccgtgccaca tccaaacatc
     3001 gaggaggtgg ctctgtccag cactggagaa atcccctttt atggcaaagc catccccatc
     3061 gagaccatca agggggggag gcacctcatt ttctgccatt ccaagaagaa atgtgatgag
     3121 ctcgccgcga agctgtccgg cctcggactc aatgctgtag catattaccg gggccttgat
     3181 gtatccgtca taccaactag cggagacgtc attgtcgtag caacggacgc tctaatgacg
     3241 ggctttaccg gtgacttcga ctcagtgatc gactgcaata catgtgtcac ccagacagtc
     3301 gacttcagcc tggacccgac cttcaccatt gagacgacga ccgtgccaca agacgcggtg
     3361 tcacgctcgc agcggcgagg caggactggt aggggcagga tgggcattta caggtttgtg
     3421 actccaggag aacggccctc gggcatgttc gattcctcgg ttctgtgcga gtgctatgac
     3481 gcgggctgtg cttggtacga gctcacgccc gccgagacct cagttaggtt gcgggcttac
     3541 ctaaacacac cagggttgcc cgtctgccag gaccatctgg agttctggga gagcgtcttt
     3601 acaggcctca cccacataga cgcccatttc ttgtcccaga ctaagcaggc aggagacaac
     3661 ttcccctacc tggtagcata ccaggctacg gtgtgcgcca gggctcaggc tccacctcca
     3721 tcgtgggacc aaatgtggaa gtgtctcata cggctaaagc ctacgctgca cgggccaacg
     3781 cccctgctgt ataggctggg agccgttcaa aacgaggtta ctaccacaca ccccataacc
     3841 aaatacatca tggcatgcat gtcggctgac ctggaggtcg tcacgagcac ctgggtgctg
     3901 gtaggcggag tcctagcagc tctggccgcg tattgcctga caacaggcag cgtggtcatt
     3961 gtgggcagga tcatcttgtc cggaaagccg gccatcattc ccgacaggga agtcctttac
     4021 cgggagttcg atgaaatgga agagtgctga gcggccgcgc cggcgggccc gaattctacc
     4081 gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg
     4141 gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc
     4201 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
     4261 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
     4321 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
     4381 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg
     4441 gtgggtccgg gggngctcag gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc
     4501 ggaggcccgg cattctgcac gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc
     4561 tcatctccgg gcctttcgac ctgcagccca agcttaccat gaccgagtac aagcccacgg
     4621 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     4681 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     4741 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     4801 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     4861 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     4921 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     4981 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     5041 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     5101 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     5161 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc
     5221 gcccgaccga aaggagcgca cgaccccatg catcggcgtc tcgagatatc agtggtccag
     5281 gctctagttt tgactcaaca atatcaccag ctgaagccta tagagtacga gccatagata
     5341 aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg
     5401 tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaaatac ataactgaga
     5461 atagagaagt tcagatcaag gtcaggaaca gatggaacag ggtcgaccct agagaaccat
     5521 cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc
     5581 aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc
     5641 ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg
     5701 tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg
     5761 tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg ctcgtccggg
     5821 atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc tggctgcctc
     5881 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     5941 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     6001 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtagatccgg
     6061 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     6121 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     6181 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     6241 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     6301 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     6361 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agcttactgg cttaactatg
     6421 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
     6481 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     6541 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6601 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6661 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6721 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6781 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6841 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6901 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6961 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     7021 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     7081 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     7141 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     7201 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     7261 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     7321 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     7381 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7441 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7501 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7561 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7621 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7681 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7741 gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta
     7801 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7861 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7921 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7981 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     8041 gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt
     8101 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     8161 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     8221 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     8281 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     8341 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     8401 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     8461 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat
     8521 tagcttggcc attgcatacg ttgtatccat atcataatat gtacatttat attggctcat
     8581 gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag taatcaatta
     8641 cggggtcatt ag
//
