LOCUS       sequence-251734-        3135 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Variant of pKD13. Lambda Red recombineering PCR template plasmid 
            carrying an F3-aadA-F3 cassette orthogonal to FRT-KanR-FRT in pKD13.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3135)
  AUTHORS   Wu Y, Bell A, Thomas GH, Bolam DN, Sargent F, Juge N, Palmer T, 
            Severi E
  TITLE     Characterisation of anhydro-sialic acid transporters from 
            mucosa-associated bacteria.
  JOURNAL   Microbiology (Reading). 2024 Mar;170(3). doi: 10.1099/mic.0.001448.
  PUBMED    38488830
REFERENCE   2  (bases 1 to 3135)
  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: 
            "Microbiology (Reading)."; date: "2024-03"; volume: "170(3). doi"; 
            pages: " 10.1099/mic.0.001448"
COMMENT     Sequence Label: pES134
FEATURES             Location/Qualifiers
     source          1..3135
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     RBS             13..24
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             31..822
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     protein_bind    823..856
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     terminator      complement(934..961)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1053..1139)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        1229..1333
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1334..1402,1403..2194)
                     /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(1552..1571)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2296..2684
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     terminator      complement(2745..2991)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     protein_bind    3071..3104
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     promoter        join(3105..3135,1..4)
                     /label=BBa_J23116
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.16"
ORIGIN
        1 tagctactag agaaagagga gaaatactaa atgagggaag cggtgatcgc cgaagtatcg
       61 actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc
      121 gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg
      181 ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt
      241 ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt
      301 gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga
      361 gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat
      421 ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg
      481 gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta
      541 acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg
      601 tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac
      661 tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct
      721 tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc
      781 cactacgtga aaggcgagat caccaaggta gtcggcaaat aagaagttcc tattcttcaa
      841 atagtatagg aacttcgaag cagctccagc ctacacaatc gctcaagacg tgtaatgctg
      901 caatctgcat gcaagcttgg cactggccac gcaaaaaggc catccgtcag gatggccttc
      961 tgcttaattt gatgcctggc agtttatggc gggcgtcctg cccgccaccc tccgggccgt
     1021 tgcttcgcaa cgttcaaatc cgctcccggc ggatttgtcc tactcaggag agcgttcacc
     1081 gacaaacaac agataaaacg aaaggcccag tctttcgact gagcctttcg ttttatttga
     1141 tgcctggcag ttccctactc tcgcatgggg agaccccaca ctaccatcgg gggccatcga
     1201 tgcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1261 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     1321 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     1381 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     1441 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     1501 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     1561 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     1621 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     1681 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     1741 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     1801 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     1861 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     1921 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     1981 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2041 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2101 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     2161 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     2221 actttagatt gatttaaaac ttcattttta atttttgcgg ccgcaagatc cggccacgat
     2281 gcgtccggcg tagaggatct gaagatcagc agttcaacct gttgatagta cgtactaagc
     2341 tctcatgttt cacgtactaa gctctcatgt ttaacgtact aagctctcat gtttaacgaa
     2401 ctaaaccctc atggctaacg tactaagctc tcatggctaa cgtactaagc tctcatgttt
     2461 cacgtactaa gctctcatgt ttgaacaata aaattaatat aaatcagcaa cttaaatagc
     2521 ctctaaggtt ttaagtttta taagaaaaaa aagaatatat aaggctttta aagcttttaa
     2581 ggtttaacgg ttgtggacaa caagccaggg atgtaacgca ctgagaagcc cttagagcct
     2641 ctcaaagcaa ttttgagtga cacaggaaca cttaacggct gacatgggaa ttagccatgg
     2701 caaagcctcg caatccagtg caaagctagc ttcttcgtct gtttctactg gtattggcac
     2761 aaacctgatt ccaatttgag caaggctatg tgccatctcg atactcgttc ttaactcaac
     2821 agaagatgct ttgtgcatac agcccctcgt ttattattta tctcctcagc cagccgctgt
     2881 gctttcagtg gatttcggat aacagaaagg ccgggaaata cccagcctcg ctttgtaacg
     2941 gagtagagac gaaagtgatt gcgcctaccc ggatattatc gtgaggatgc gtcatcgcca
     3001 ttaattcact gatcagtgat aagctgtcaa acatgagaat taattccggg gatccgtcga
     3061 cctgcagttc gaagttccta ttcttcaaat agtataggaa cttcttgaca gctagctcag
     3121 tcctagggac tatgc
//
