LOCUS       sequence-216989-        8272 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BTK assembled plasmid- broad-host-range GFP expression from PA1 
            promoter (spectinomycin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8272)
  AUTHORS   Leonard SP, Perutka J, Powell JE, Geng P, Richhart DD, Byrom M, Kar 
            S, Davies BW, Ellington AD, Moran NA, Barrick JE
  TITLE     Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit 
            for Modular Assembly of Broad-Host-Range Plasmids.
  JOURNAL   ACS Synth Biol. 2018 May 18;7(5):1279-1290. doi: 
            10.1021/acssynbio.7b00399. Epub 2018 Apr 13.
  PUBMED    29608282
REFERENCE   2  (bases 1 to 8272)
  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"; doi: 
            "10.1021/acssynbio.7b00399"; journalName: "ACS Synth Biol"; date: 
            "2018-05-18- 18"; volume: "7"; issue: "5"; pages: "1279-1290"
COMMENT     Sequence Label: pBTK520
FEATURES             Location/Qualifiers
     source          1..8272
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             913..1752
                     /codon_start=1
                     /gene="repA"
                     /product="replication protein A of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepA
                     /translation="MATHKPINILEAFAAAPPPLDYVLPNMVAGTVGALVSPGGAGKSM
                     LALQLAAQIAGGPDLLEVGELPTGPVIYLPAEDPPTAIHHRLHALGAHLSAEERQAVAD
                     GLLIQPLIGSLPNIMAPEWFDGLKRAAEGRRLMVLDTLRRFHIEEENASGPMAQVIGRM
                     EAIAADTGCSIVFLHHASKGAAMMGAGDQQQASRGSSVLVDNIRWQSYLSSMTSAEAEE
                     WGVDDDQRRFFVRFGVSKANYGAPFADRWFRRHDGGVLKPAVLERQRKSKGVPRGEA"
     CDS             1739..2590
                     /codon_start=1
                     /gene="repC"
                     /product="replication protein C of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepC
                     /translation="MVKPKNKHSLSHVRHDPAHCLAPGLFRALKRGERKRSKLDVTYDY
                     GDGKRIEFSGPEPLGADDLRILQGLVAMAGPNGLVLGPEPKTEGGRQLRLFLEPKWEAV
                     TADAMVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
                     YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
                     TGKASIDTLCGYVWPSEASGSTMRKRRQRVREALPELVALGWTVTEFAAGKYDITRPKA
                     AG"
     CDS             2970..3761
                     /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"
     terminator      complement(3780..3807)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(3823..3894)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    4910..4934
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     rep_origin      5641..6035
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
     oriT            6377..6464
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     CDS             join(7703..8272,1..402)
                     /codon_start=1
                     /gene="repB"
                     /product="replication protein B of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepB
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDATTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
ORIGIN
        1 gagcaggccc agcggcagca ggagaaggcc cgcaggctgg ccagcctcga actgcccgag
       61 cggcagctta gccgccaccg gcgcacggcg ctggacgagt accgcagcga gatggccggg
      121 ctggtcaagc gcttcggtga tgacctcagc aagtgcgact ttatcgccgc gcagaagctg
      181 gccagccggg gccgcagtgc cgaggaaatc ggcaaggcca tggccgaggc cagcccagcg
      241 ctggcagagc gcaagcccgg ccacgaagcg gattacatcg agcgcaccgt cagcaaggtc
      301 atgggtctgc ccagcgtcca gcttgcgcgg gccgagctgg cacgggcacc ggcaccccgc
      361 cagcgaggca tggacagggg cgggccagat ttcagcatgt agtgcttgcg ttggtactca
      421 cgcctgttat actatgagta ctcacgcaca gaagggggtt ttatggaata cgaaaaaagc
      481 gcttcagggt cggtctacct gatcaaaagt gacaagggct attggttgcc cggtggcttt
      541 ggttatacgt caaacaaggc cgaggctggc cgcttttcag tcgctgatat ggccagcctt
      601 aaccttgacg gctgcacctt gtccttgttc cgcgaagaca agcctttcgg ccccggcaag
      661 tttctcggtg actgatatga aagaccaaaa ggacaagcag accggcgacc tgctggccag
      721 ccctgacgct gtacgccaag cgcgatatgc cgagcgcatg aaggccaaag ggatgcgtca
      781 gcgcaagttc tggctgaccg acgacgaata cgaggcgctg cgcgagtgcc tggaagaact
      841 cagagcggcg cagggcgggg gtagtgaccc cgccagcgcc taaccaccaa ctgcctgcaa
      901 aggaggcaat caatggctac ccataagcct atcaatattc tggaggcgtt cgcagcagcg
      961 ccgccaccgc tggactacgt tttgcccaac atggtggccg gtacggtcgg ggcgctggtg
     1021 tcgcccggtg gtgccggtaa atccatgctg gccctgcaac tggccgcaca gattgcaggc
     1081 gggccggatc tgctggaggt gggcgaactg cccaccggcc cggtgatcta cctgcccgcc
     1141 gaagacccgc ccaccgccat tcatcaccgc ctgcacgccc ttggggcgca cctcagcgcc
     1201 gaggaacggc aagccgtggc tgacggcctg ctgatccagc cgctgatcgg cagcctgccc
     1261 aacatcatgg ccccggagtg gttcgacggc ctcaagcgcg ccgccgaggg ccgccgcctg
     1321 atggtgctgg acacgctgcg ccggttccac atcgaggaag aaaacgccag cggccccatg
     1381 gcccaggtca tcggtcgcat ggaggccatc gccgccgata ccgggtgctc tatcgtgttc
     1441 ctgcaccatg ccagcaaggg cgcggccatg atgggcgcag gcgaccagca gcaggccagc
     1501 cggggcagct cggtactggt cgataacatc cgctggcagt cctacctgtc gagcatgacc
     1561 agcgccgagg ccgaggaatg gggtgtggac gacgaccagc gccggttctt cgtccgcttc
     1621 ggtgtgagca aggccaacta tggcgcaccg ttcgctgatc ggtggttcag gcggcatgac
     1681 ggcggggtgc tcaagcccgc cgtgctggag aggcagcgca agagcaaggg ggtgccccgt
     1741 ggtgaagcct aagaacaagc acagcctcag ccacgtccgg cacgacccgg cgcactgtct
     1801 ggcccccggc ctgttccgtg ccctcaagcg gggcgagcgc aagcgcagca agctggacgt
     1861 gacgtatgac tacggcgacg gcaagcggat cgagttcagc ggcccggagc cgctgggcgc
     1921 tgatgatctg cgcatcctgc aagggctggt ggccatggct gggcctaatg gcctagtgct
     1981 tggcccggaa cccaagaccg aaggcggacg gcagctccgg ctgttcctgg aacccaagtg
     2041 ggaggccgtc accgctgatg ccatggtggt caaaggtagc tatcgggcgc tggcaaagga
     2101 aatcggggca gaggtcgata gtggtggggc gctcaagcac atacaggact gcatcgagcg
     2161 cctttggaag gtatccatca tcgcccagaa tggccgcaag cggcaggggt ttcggctgct
     2221 gtcggagtac gccagcgacg aggcggacgg gcgcctgtac gtggccctga accccttgat
     2281 cgcgcaggcc gtcatgggtg gcggccagca tgtgcgcatc agcatggacg aggtgcgggc
     2341 gctggacagc gaaaccgccc gcctgctgca ccagcggctg tgtggctgga tcgaccccgg
     2401 caaaaccggc aaggcttcca tagatacctt gtgcggctat gtctggccgt cagaggccag
     2461 tggttcgacc atgcgcaagc gccgccagcg ggtgcgcgag gcgttgccgg agctggtcgc
     2521 gctgggctgg acggtaaccg agttcgcggc gggcaagtac gacatcaccc ggcccaaggc
     2581 ggcaggctga ctcacgttaa gggattttgg tcatgaccag ccaggacaga aatgcctcga
     2641 cttcgctgct acccaaggtt gccgggtgac gcacaccgtg gaaacggatg aaggcacgaa
     2701 cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt atgcgctcac
     2761 gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg gcggttttca
     2821 tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc aagcagcaag
     2881 cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt acgcagcagg
     2941 gcagtcgccc taaaacaaag ttaaacatta tgagggaagc ggtgatcgcc gaagtatcga
     3001 ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg
     3061 tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat attgatttgc
     3121 tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt
     3181 tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg
     3241 ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg caatttggag
     3301 aatggcagcg caatgacatt cttgcgggta tcttcgagcc agccacgatc gacattgatc
     3361 tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt ccagcggcgg
     3421 aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat gaaaccttaa
     3481 cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg cttacgttgt
     3541 cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact
     3601 gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct agacaggctt
     3661 atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc
     3721 actacgtgaa aggcgagatc accaaggtag tcggcaaata agcggccgct cggtataaac
     3781 gcagaaaggc ccacccgaag gtgagccagt gtgactctag tagagagcgt tcaccgacaa
     3841 acaacagata aaacgaaagg cccagtcttt cgactgagcc tttcgtttta tttgatgcct
     3901 ggtgtactgc agtgcactag ttaactgcct tgatctgtcg ggtgtctgct tctctcacag
     3961 gcttaggtgg atctgctgtc cccagtcagt gcggtcatca tgcagtcatc cgagcgtgta
     4021 ttgcacacat ttgtttggcg catgatgagc cgtgatgacc ccgtcgtctc attggcagct
     4081 gacaaatgct ctttccctaa actcccccca taaaaaaacc cgccgaagcg ggtttttacg
     4141 ttatttgcgg attaacgatt acggatttat ttgtatagtt catccatgcc gtgtgtaatc
     4201 ccagcagccg tgacaaactc aagaaggacc atgtggtcgc gcttttcgtt gggatctttc
     4261 gaaagggcag attgagtgga caggtaatgg ttgtctggta acagcacagg cccatcgcca
     4321 attggggtgt tttgttggta atggtcggct agttgaacgc ttccatcctc aatgttgtgt
     4381 ctgatcttga agttaacctt gataccgttc ttttgcttgt cggccataat gtacacatta
     4441 tgggagttat agttatattc cagtttgtgg cctaaaatat ttccatcttc cttaaagtca
     4501 atacccttta actcgattct attaacaagg gtatcaccct cgaacttcac ttcggcgcgt
     4561 gtcttatagt tgccgtcatc cttaaaaaag atggttctct cctgtacata gccttcgggc
     4621 attgcactct tgaaaaagtc gtgctgtttc atgtgatctg ggtagcgcgc aaagcactga
     4681 accccatacc cgaacgtagt gaccagtgtt ggccatggaa caggtagttt acccgtggtg
     4741 caaataaatt taagggtcag cttgccatat gttgcgtcgc cttcaccctc tccactgaca
     4801 gagaacttgt gcccattaac atcaccgtct aattccacga ggattgggac aactcctgtg
     4861 aaaagctctt ctcctttact catatgtaat atctcctctg tttctgtatg gaattgttat
     4921 ccgctcacaa ttcctcgccg tgtccctctc gatggctgta agtatcctat aggttagact
     4981 ttaagtcaat actctttttg ataacgttcg tctgcaatta tcggccagtt cagtgagacg
     5041 gtattgcgac gaattgccac gttgtcgcag tgtctgtact gtgggttaat gtctgtgatc
     5101 gccgtcgtga gttcgtgtca tccgcgcatg attcagatgg tcctggagat cgttgcttgt
     5161 accggctgtt tgtggctcta ccatctagat gcgaattcag gggcctgtca gaccaagttt
     5221 actcatatat actttagatt gatttctgaa agcgaccagg tgctcggcgt ggcaagactc
     5281 gcagcgaacc cgtagaaagc catgctccag ccgcccgcat tggagaaatt cttcaaattc
     5341 ccgttgcaca tagcccggca attcctttcc ctgctctgcc ataagcgcag cgaatgccgg
     5401 gtaatactcg tcaacgatct gatagagaag ggtttgctcg ggtcggtggc tctggtaacg
     5461 accagtatcc cgatcccggc tggccgtcct ggccgccaca tgaggcatgt tccgcgtcct
     5521 tgcaatactg tgtttacata cagtctatcg cttagcggaa agttctttta ccctcagccg
     5581 aaatgcctgc cgttgctaga cattgccagc cagtgcccgt cactcccgta ctaactgtca
     5641 cgaacccctg caataactgt cacgcccccc tgcaataact gtcacgaacc cctgcaataa
     5701 ctgtcacgcc cccaaacctg caaacccagc aggggcgggg gctggcgggg tgttggaaaa
     5761 atccatccat gattatctaa gaataatcca ctaggcgcgg ttatcagcgc ccttgtgggg
     5821 cgctgctgcc cttgcccaat atgcccggcc agaggccgga tagctggtct attcgctgcg
     5881 ctaggctaca caccgcccca ccgctgcgcg gcagggggaa aggcgggcaa agcccgctaa
     5941 accccacacc aaaccccgca gaaatacgct ggagcgcttt tagccgcttt agcggccttt
     6001 ccccctaccc gaagggtggg ggcgcgtgtg cagccccgca gggcctgtct cggtcgatca
     6061 ttcagcccgg ctcatccttc tggcgtggcg gcagaccgaa caaggcgcgg tcgtggtcgc
     6121 gttcaaggta cgcatccatt gccgccatga gccgatcctc cggccactcg ctgctgttca
     6181 ccttggccaa aatcatggcc cccaccagca ccttgcgcct tgtttcgttc ttgcgctctt
     6241 gctgctgttc ccttgcccgc acccgctgaa tttcggcatt gattcgcgct cgttgttctt
     6301 cgagcttggc cagccgatcc gccgccttgt tgctcccctt aaccatcttg acaccccatt
     6361 gttaatgtgc tgtctcgtag gctatcatgg aggcacagcg gcggcaatcc cgaccctact
     6421 ttgtagggga gggcgcactt accggtttct cttcgagaaa ctggcctaac ggccaccctt
     6481 cgggcggtgc gctctccgag ggccattgca tggagccgaa aagcaaaagc aacagcgagg
     6541 cagcatggcg atttatcacc ttacggcgaa aaccggcagc aggtcgggcg gccaatcggc
     6601 cagggccaag gccgactaca tccagcgcga aggcaagtat gcccgcgaca tggatgaagt
     6661 cttgcacgcc gaatccgggc acatgccgga gttcgtcgag cggcccgccg actactggga
     6721 tgctgccgac ctgtatgaac gcgccaatgg gcggctgttc aaggaggtcg aatttgccct
     6781 gccggtcgag ctgaccctcg accagcagaa ggcgctggcg tccgagttcg cccagcacct
     6841 gaccggtgcc gagcgcctgc cgtatacgct ggccatccat gccggtggcg gcgagaaccc
     6901 gcactgccac ctgatgatct ccgagcggat caatgacggc atcgagcggc ccgccgctca
     6961 gtggttcaag cggtacaacg gcaagacccc ggagaagggc ggggcacaga agaccgaagc
     7021 gctcaagccc aaggcatggc ttgagcagac ccgcgaggca tgggccgacc atgccaaccg
     7081 ggcattagag cgggctggcc acgacgcccg cattgaccac agaacacttg aggcgcaggg
     7141 catcgagcgc ctgcccggtg ttcacctggg gccgaacgtg gtggagatgg aaggccgggg
     7201 catccgcacc gaccgggcag acgtggccct gaacatcgac accgccaacg cccagatcat
     7261 cgacttacag gaataccggg aggcaataga ccatgaacgc aatcgacaga gtgaagaaat
     7321 ccagaggcat caacgagtta gcggagcaga tcgaaccgct ggcccagagc atggcgacac
     7381 tggccgacga agcccggcag gtcatgagcc agacccagca ggccagcgag gcgcaggcgg
     7441 cggagtggct gaaagcccag cgccagacag gggcggcatg ggtggagctg gccaaagagt
     7501 tgcgggaggt agccgccgag gtgagcagcg ccgcgcagag cgcccggagc gcgtcgcggg
     7561 ggtggcactg gaagctatgg ctaaccgtga tgctggcttc catgatgcct acggtggtgc
     7621 tgctgatcgc atcgttgctc ttgctcgacc tgacgccact gacaaccgag gacggctcga
     7681 tctggctgcg cttggtggcc cgatgaagaa cgacaggact ttgcaggcca taggccgaca
     7741 gctcaaggcc atgggctgtg agcgcttcga tatcggcgtc agggacgcca ccaccggcca
     7801 gatgatgaac cgggaatggt cagccgccga agtgctccag aacacgccat ggctcaagcg
     7861 gatgaatgcc cagggcaatg acgtgtatat caggcccgcc gagcaggagc ggcatggtct
     7921 ggtgctggtg gacgacctca gcgagtttga cctggatgac atgaaagccg agggccggga
     7981 gcctgccctg gtagtggaaa ccagcccgaa gaactatcag gcatgggtca aggtggccga
     8041 cgccgcaggc ggtgaacttc gggggcagat tgcccggacg ctggccagcg agtacgacgc
     8101 cgacccggcc agcgccgaca gccgccacta tggccgcttg gcgggcttca ccaaccgcaa
     8161 ggacaagcac accacccgcg ccggttatca gccgtgggtg ctgctgcgtg aatccaaggg
     8221 caagaccgcc accgctggcc cggcgctggt gcagcaggct ggccagcaga tc
//
