LOCUS       sequence-476750-        5319 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression Promoter (RpoD sigma factor, BT_RS06635), 
            replicates via RepA; 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 5319)
  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 5319)
  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: pMS03
FEATURES             Location/Qualifiers
     source          1..5319
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..389
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             1025..1090
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(4044..4112,4113..4904)
                     /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(4262..4281)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     oriT            5035..5144
                     /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 aaaaaaagaa tatataaggc ttttaaagct tttaaggttt aacggttgtg
      301 gacaacaagc cagggatgta acgcactgag aagcccttag agcctctcaa agcaattttg
      361 agtgacacag gaacacttaa cggctgacat gggaattccc ctccacctta ttcttcccga
      421 ccgaacgagc ggtattcaag cggagtaagc ccggtacgtt ttttgaaaaa ggagaaaaaa
      481 tattcggtag actggaagcc cagaaaaaaa gaaatttctt ttactgactg ggaagttccc
      541 accaacattt gcttggcttt acgaagtttc agttgatatc agtgatctgg aagaagcaat
      601 gaaagctgct gttaagtctc cgaatcaggt attgttcctg acaggtgtat tcccatccgg
      661 taaacgcgga tactttgcag ttgatctgac tcaggaataa attataaatt aaggtaagaa
      721 gattgtagga taagctaatg aaatagaaaa aggatgccgt cacacaactt gtcggcattc
      781 ttttttgttt tattagttga aaatatagtg aaaaagttgc ctaaatatgt atgttaacaa
      841 attatttgtc gtaactttgc actccaaatc tgtttttaaa gacccgggca tgccaggatc
      901 cgaattcgag ctcggcgcgc ctgcaggtcg acaaacttgc ggccgcataa tgcttaagtc
      961 catatggcag atctcaattg gatatcggcc ggccacgcga tcgctgacgt cggtaccctc
     1021 gagtgactac aaggaccacg acggcgacta taaggaccac gacatcgact acaaggatga
     1081 cgacgacaag tgagggccct ataatttggt gaattggagt tattttcata tttttgcatc
     1141 ccgaagagtt tctcttaaag agagaaacat cttttgcata ccttttccga ccgaattttt
     1201 atgtcgtaaa gaggggcttt gcagggggtg gactcagaaa gatgagaata gatgactatt
     1261 gtagttgaaa cacatagaaa gttgctgata tacagaccga tacgcatatc gggatgaacc
     1321 atgagtacgt tcttttctca aaaaacataa atattcgaaa agagatgcaa taaattaagg
     1381 agaggttata gcttagcatg acaaaaaaga aattgcccgt tcgttttacg ggtcagcact
     1441 ttactattga taaagtgcta ataaaagatg caataagaca agcaaatata agtaatcagg
     1501 atacggtttt agatattggg gcaggcaagg ggtttcttac tgttcattta ttaaaaatcg
     1561 ccaacaatgt tgttgctatt gaaaacgaca cagctttggt tgaacattta cgaaaattat
     1621 tttctgatgc ccgaaatgtt caagttgtcg gttgtgattt taggaatttt gcagttccga
     1681 aatttccttt caaagtggtg tcaaatattc cttatggcat tacttccgat attttcaaaa
     1741 tcctgatgtt tgagagtctt ggaaattttc tgggaggttc cattgtcctt caattagaac
     1801 ctacacaaaa gttattttcg aggaagcttt acaatccata taccgttttc tatcatactt
     1861 tttttgattt gaaacttgtc tatgaggtag gtcctgaaag tttcttgcca ccgccaactg
     1921 tcaaatcagc cctgttaaac attaaaagaa aacacttatt ttttgatttt aagtttaaag
     1981 ccaaatactt agcatttatt tcctatctgt tagagaaacc tgatttatct gtaaaaacag
     2041 ctttaaagtc gattttcagg aaaagtcagg tcaggtcaat ttcggaaaaa ttcggtttaa
     2101 accttaatgc tcaaattgtt tgtttgtctc caagtcaatg gttaaactgt tttttggaaa
     2161 tgctggaagt tgtccctgaa aaatttcatc cttcgtaggc atgcggcgct ccccgaccga
     2221 tgatttttaa gtgactgatt ttgtgctgtt ttgggggtat attaagaata aaagaaatag
     2281 aataagttaa gtacttgata cacaatatag ggcattttcc atattggaaa ttctcatttt
     2341 ccaatctaga aaataccgat ttcctaatat gatactaaat aggaaaataa tatttccctt
     2401 aatattgttt ttatggaaaa taatagttta ctttgtggag aataatattt cccaaaaaca
     2461 tatcaaaatg gaaaataaaa aagcagttaa gttaaccgat tttcaaaaga acgaagaaaa
     2521 tccttttatg aaacaagcta tagaaggtat tgaaaatcat gttgttaaaa agtataagag
     2581 taatagtggt ggcgataaga gagctgtagt agctttagcc gacactgaaa ctggagaagt
     2641 gtttaagact tcgtttatcc gtcaaataga agtagatgaa gaacaattca ctaaattgta
     2701 tctttctaac tttgctgcat tctttgacct atcacaagca gctattcggg tttttggtta
     2761 ctttatgacc tgcatgaaac ccaaaaatga tttaatcatt ttcaatagaa aaaaatgcct
     2821 agaatatacc aaatacaaaa cagacaaagc cgtttataaa ggacttgcag aacttgtaaa
     2881 agctgaaatc atagcccgag gaccagccga taatctttgg tttattaatc ctctgatagt
     2941 attcaatggt gaccgagtga catttgctaa aacatacgtt cggaaaaaga ctttagctgc
     3001 ccaaaagaaa gaagaagcag agaaacgaca attatcactt ggctttgatg aacagtaaca
     3061 ctccattgag tgaagctgcc gtttggtcgc tccccctttg ggggcggggg gggatagata
     3121 aagttcctct atgtaaagtt ataatggggg atgaaaggca aggttcgcta accttacccc
     3181 gattgtttat gcttcccagc attcaatcta tccccgttta tgggtttctc tttcacccta
     3241 caaagataac ctcatggggg aaaaatgtcc aagaatatgg ggtaaaaact atcaagtcgg
     3301 tagaaaataa gtatctttga agtacatttt tagtagaggt actggtgtgc ctaggcgaag
     3361 cagataggca aaaataaagc cgctggagat gtcggatgtc cgtcaattaa ctccagcggc
     3421 ttgtattttg gtaactctcc cgtatcggtc agctaccgtt tggtcagcaa caaaggtagt
     3481 actttattat cgcaaatcca aatgtttttc cctttatttg tgcatttaca aaacattcac
     3541 taacttcgca cctatgcagt atggtgatag cacactagca ccctaaagag tgcgtgtgta
     3601 aggggacaag cccccttaac cccctgtcag ccggctgtca ccggctgtta tcgattgtca
     3661 caaaaatgtt aacttatggg agcaacaagt attcatgtac aagcagtgaa gccggggagc
     3721 gagattcaca actttaggga aaaagagttg gactatgttc gtcccgaact tagtcatttg
     3781 aatgaaagct gggttggaga tagcatttcc catcggctgg agagtgcaaa acaaagatgc
     3841 tagcatatag cgactttttt acaaaatgct aaaatttgtt gatttccgtc aagcaattgt
     3901 tgagcaaaaa tgtcttttac gataaaatga tacctcaata tcaactgttt agcaaaacga
     3961 tatttctctt aaagagagaa acaccttttt gttcaccaat ccccgacttt taatcccgcg
     4021 gccatgattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     4081 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     4141 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     4201 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     4261 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     4321 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     4381 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     4441 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     4501 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     4561 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4621 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4681 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4741 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4801 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4861 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4921 actcataacg cgtcaattcg agggggatca attccgtgat aggtgggctg cccttcctgg
     4981 ttggcttggt ttcatcagcc atccgcttgc cctcatctgt tacgccggcg gtagccggcc
     5041 agcctcgcag agcaggattc ccgttgagca ccgccaggtg cgaataaggg acagtgaaga
     5101 aggaacaccc gctcgcgggt gggcctactt cacctatcct gctctgcgag gctgacgccg
     5161 ttggatacac caaggaaagt ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa
     5221 aaaggatgga tataccgaaa aaatcgctat aatgaccccg aagcagggtt atgcagcgga
     5281 aaacggaatt gatccggcca cgatgcgtcc ggcgtagag
//
