LOCUS       sequence-475921-        5967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible Promoter (anhydrotetracycline-induced, tetO/tetR system), 
            integrates via IntN1; ermF, C-terminal 3x-FLAG, R6K ori, RP4 oriT.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5967)
  AUTHORS   Schnizlein MK, Dubey AA, Fiebig A, Crosson S
  TITLE     Genetic- and culture-based tools for studying Bacteroides fragilis.
  JOURNAL   Microbiol Resour Announc. 2025 May 8;14(5):e0000625. doi: 
            10.1128/mra.00006-25. Epub 2025 Mar 25.
  PUBMED    40130927
REFERENCE   2  (bases 1 to 5967)
  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: "Microbiol 
            Resour Announc."; date: "2025-05-8"; volume: "14(5):e0000625. doi"; 
            pages: " 10.1128/mra.00006-25. Epub 2025 Mar 25"
COMMENT     Sequence Label: pMS04
FEATURES             Location/Qualifiers
     source          1..5967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..390
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(420..1043)
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     951..970
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     protein_bind    1314..1332
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1376..1394
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             1567..1632
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(4697..4765,4766..5557)
                     /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(4915..4934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     oriT            5688..5797
                     /note="incP origin of transfer"
ORIGIN
        1 gatctgaaga tcagcagttc aacctgttga tagtacgtac taagctctca tgtttcacgt
       61 actaagctct catgtttaac gtactaagct ctcatgttta acgaactaaa ccctcatggc
      121 taacgtacta agctctcatg gctaacgtac taagctctca tgtttcacgt actaagctct
      181 catgtttgaa caataaaatt aatataaatc agcaacttaa atagcctcta aggttttaag
      241 ttttataaga aaaaaaaaga atatataagg cttttaaagc ttttaaggtt taacggttgt
      301 ggacaacaag ccagggatgt aacgcactga gaagccctta gagcctctca aagcaatttt
      361 gagtgacaca ggaacactta acggctgaca tgggaattct tcccctccac cgcggtggct
      421 taagacccac tttcacattt aagttgtttt tctaatccgc atatgatcaa ttcaaggccg
      481 aataagaagg ctggctctgc accttggtga tcaaataatt cgatagcttg tcgtaataat
      541 ggcggcatac tatcagtagt aggtgtttcc ctttcttctt tagcgacttg atgctcttga
      601 tcttccaata cgcaacctaa agtaaaatgc cccacagcgc tgagtgcata taatgcattc
      661 tctagtgaaa aaccttgttg gcataaaaag gctaattgat tttcgagagt ttcatactgt
      721 ttttctgtag gccgtgtacc taaatgtact tttgctccat cgcgatgact tagtaaagca
      781 catctaaaac ttttagcgtt attacgtaaa aaatcttgcc agctttcccc ttctaaaggg
      841 caaaagtgag tatggtgcct atctaacatc tcaatggcta aggcgtcgag caaagcccgc
      901 ttatttttta catgccaata caatgtaggc tgctctacac ctagcttctg ggcgagttta
      961 cgggttgtta aaccttcgat tccgacctca ttaagcagct ctaatgcgct gttaatcact
     1021 ttacttttat ctaatctaga catattcgtt taatatcata aataatttat tttattttaa
     1081 aatgcgcggg tgcaaaggta agaggtttta ttttaactac caaatgtttt cggaagtttt
     1141 ttcgcttttc tttttctatc gtttctcaga ctctcttagc gaaagggaaa gaaggtaaag
     1201 aagaaaaaca aaacgccttt tctttttttg cacccgcttt ccaagagaag aaagccttgt
     1261 taaattgact tagtgtaaaa gcgcagtact gcttgaccat aagaacaaaa aaatctctat
     1321 cactgatagg gataaagttt ggaagataaa gctaaaagtt cttatctttg cagtctccct
     1381 atcagtgata gagacgaaat aaagacatat aaaagaaaag acaccccggg catgccagga
     1441 tccgaattcg agctcggcgc gcctgcaggt cgacaagctt gcggccgcat aatgcttaag
     1501 tccatatggc agatctcaat tggatatcgg ccggccacgc gatcgctgac gtcggtaccc
     1561 tcgagtgact acaaggacca cgacggcgac tataaggacc acgacatcga ctacaaggat
     1621 gacgacgaca agtgagggcc ctataatttg gtgaattgga gttattttca tatttttgca
     1681 tcccgaagag tttctcttaa agagagaaac atcttttgca taccttttcc gaccgaattt
     1741 ttatgtcgta aagaggggct ttgcaggggg tggactcaga aagatgagaa tagatgacta
     1801 ttgtagttga aacacataga aagttgctga tatacagacc gatacgcata tcgggatgaa
     1861 ccatgagtac gttcttttct caaaaaacat aaatattcga aaagagatgc aataaattaa
     1921 ggagaggtta tagcttagca tgacaaaaaa gaaattgccc gttcgtttta cgggtcagca
     1981 ctttactatt gataaagtgc taataaaaga tgcaataaga caagcaaata taagtaatca
     2041 ggatacggtt ttagatattg gggcaggcaa ggggtttctt actgttcatt tattaaaaat
     2101 cgccaacaat gttgttgcta ttgaaaacga cacagctttg gttgaacatt tacgaaaatt
     2161 attttctgat gcccgaaatg ttcaagttgt cggttgtgat tttaggaatt ttgcagttcc
     2221 gaaatttcct ttcaaagtgg tgtcaaatat tccttatggc attacttccg atattttcaa
     2281 aatcctgatg tttgagagtc ttggaaattt tctgggaggt tccattgtcc ttcaattaga
     2341 acctacacaa aagttatttt cgaggaagct ttacaatcca tataccgttt tctatcatac
     2401 tttttttgat ttgaaacttg tctatgaggt aggtcctgaa agtttcttgc caccgccaac
     2461 tgtcaaatca gccctgttaa acattaaaag aaaacactta ttttttgatt ttaagtttaa
     2521 agccaaatac ttagcattta tttcctatct gttagagaaa cctgatttat ctgtaaaaac
     2581 agctttaaag tcgattttca ggaaaagtca ggtcaggtca atttcggaaa aattcggttt
     2641 aaaccttaat gctcaaattg tttgtttgtc tccaagtcaa tggttaaact gttttttgga
     2701 aatgctggaa gttgtccctg aaaaatttca tccttcgtag gcatgcctca tcggattctt
     2761 ttaatatcgc tactctctac gttcaaccaa aagaaataat gacttactgc tatatttttt
     2821 gcacgtgtgg ggaaaatgtg gggaaaattc aagcaaaaga aaaagctaag tattgaacta
     2881 tcaaatactt agctttcttt cttgtaccca gaccccgcat ttgaaataat taaagtgggg
     2941 aaaatgtggg taaaaagaaa aatgcggaaa aacgccacaa ttacactgta tttcaatatg
     3001 ttataatcct attaaatttt aatccaagtt taatcgaatt gcaaaatatt tagcagatgt
     3061 ggggaaaatg ctggggaaaa tatttatatt tgcagcagag taaaattcaa cctgaaagtc
     3121 tatgaaagta acctttatca ttaaaaaagc agccaaacga tatgatacag aatccatggc
     3181 tacaatctat gtccgtttta gaaacggaag gcagttagac tccgttgctc ctactcagtt
     3241 agccatcaat cccaatctat gggatgataa agacgaatgt gtaaaaacga aagctgtctg
     3301 caatgaagaa atgcgtaccc atataaatga agagatacgc cagttgaaaa cctatatcga
     3361 gaaggtatat caacaagaaa aggaagcaat agacaaagaa tggctaaaaa caacacttga
     3421 taaattttac catcctgaaa aatatttttt gccggaggaa gtggttatca agcctaccat
     3481 tggagaacta ttcgatgaat ttctaaacaa gcaccctttg tcggaagtac gaaagaaaaa
     3541 tttccgggtt gtcaaaagag ccttactgcg ttatgaacta tatgtaaggg ctacaaagag
     3601 aggacaaaag ggctttatcc ttgatgtgga tttggtaaca cctgacacgc ttcgggatat
     3661 gtgggatttc tttcagaacg aataccagta ttatgaactt tacccgagca tttatgaagc
     3721 cattcccgaa aagaggacac cacagcccag aagcaaaaac acgctgatag actgtttttc
     3781 aagaatacgc acattcttcc tgtggtgctt cgataacaaa cgcaccacaa acagaccttt
     3841 cgacaagttt ccgatagagg agtgtacata tggtacacct tattatataa cactcgaaga
     3901 aagggacagg atttttaatg cagacctttc tgccacccca caactggcaa tacagaggga
     3961 tatattcata tttcagacac tgataggatg cagggtgagc gacctgtacc gaatgaccaa
     4021 actaaatgtg gtcaatgaag ccatagaata tattcccaag aaaaccaaag aggggaatcc
     4081 ggttacggta cgtgttccac ttaacgacaa agcgaaagaa atccttgaac gctacaaaga
     4141 atatgaggga aaactgttgc cgttcatatc cgagcaaaag tacaatgatg ccataaaaaa
     4201 gatattcaaa ttagctggag ttgaccgcat cgtaacaatc ttagacccgt tgacgcacaa
     4261 cgaaatcaaa cgacctattt atgaagtggc aagcagccat ctggcaagac gtacgtttat
     4321 cggcaatatc tataaaaaag tgaaagaccc gaaccttgtt tccgcactgt cgggacacaa
     4381 ggagggaagc aaagctttca gacgatacag ggatattgac gaagaaatga agaaagacct
     4441 tgtaaaacta ctggactgat aaaagtatca aacagtttca tagatagtta ggctagcata
     4501 tagcgacttt tttacaaaat gctaaaattt gttgatttcc gtcaagcaat tgttgagcaa
     4561 aaatgtcttt tacgataaaa tgatacctca atatcaactg tttagcaaaa cgatatttct
     4621 cttaaagaga gaaacacctt tttgttcacc aatccccgac ttttaatccc gcggccatga
     4681 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     4741 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     4801 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     4861 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4921 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4981 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     5041 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     5101 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     5161 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     5221 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     5281 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     5341 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     5401 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     5461 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     5521 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     5581 acgcgtcaat tcgaggggga tcaattccgt gataggtggg ctgcccttcc tggttggctt
     5641 ggtttcatca gccatccgct tgccctcatc tgttacgccg gcggtagccg gccagcctcg
     5701 cagagcagga ttcccgttga gcaccgccag gtgcgaataa gggacagtga agaaggaaca
     5761 cccgctcgcg ggtgggccta cttcacctat cctgcccggc tgacgccgtt ggatacacca
     5821 aggaaagtct acacgaaccc tttggcaaaa tcctgtatat cgtgcgaaaa aggatggata
     5881 taccgaaaaa atcgctataa tgaccccgaa gcagggttat gcagcggaaa acggaattga
     5941 tccggccacg atgcgtccgg cgtagag
//
