LOCUS       sequence-258167-        8118 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BTK assembled plasmid- broad-host-range GFP expression from PA1 
            promoter (kanamycin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8118)
  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 8118)
  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: pBTK519
FEATURES             Location/Qualifiers
     source          1..8118
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            391..478
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     rep_origin      820..1214
                     /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)"
     protein_bind    1921..1945
                     /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)."
     terminator      2961..3032
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3048..3075
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(3121..3936)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(3121..3936)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3844..3863
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3928..3947
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(4111..4962)
                     /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             complement(4949..5788)
                     /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             complement(6299..7270)
                     /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 gctcggcacc ggtcaggtgc tgggcgaact cggacgccag cgccttctgc tggtcgaggg
       61 tcagctcgac cggcagggca aattcgacct ccttgaacag ccgcccattg gcgcgttcat
      121 acaggtcggc agcatcccag tagtcggcgg gccgctcgac gaactccggc atgtgcccgg
      181 attcggcgtg caagacttca tccatgtcgc gggcatactt gccttcgcgc tggatgtagt
      241 cggccttggc cctggccgat tggccgcccg acctgctgcc ggttttcgcc gtaaggtgat
      301 aaatcgccat gctgcctcgc tgttgctttt gcttttcggc tccatgcaat ggccctcgga
      361 gagcgcaccg cccgaagggt ggccgttagg ccagtttctc gaagagaaac cggtaagtgc
      421 gccctcccct acaaagtagg gtcgggattg ccgccgctgt gcctccatga tagcctacga
      481 gacagcacat taacaatggg gtgtcaagat ggttaagggg agcaacaagg cggcggatcg
      541 gctggccaag ctcgaagaac aacgagcgcg aatcaatgcc gaaattcagc gggtgcgggc
      601 aagggaacag cagcaagagc gcaagaacga aacaaggcgc aaggtgctgg tgggggccat
      661 gattttggcc aaggtgaaca gcagcgagtg gccggaggat cggctcatgg cggcaatgga
      721 tgcgtacctt gaacgcgacc acgaccgcgc cttgttcggt ctgccgccac gccagaagga
      781 tgagccgggc tgaatgatcg accgagacag gccctgcggg gctgcacacg cgcccccacc
      841 cttcgggtag ggggaaaggc cgctaaagcg gctaaaagcg ctccagcgta tttctgcggg
      901 gtttggtgtg gggtttagcg ggctttgccc gcctttcccc ctgccgcgca gcggtggggc
      961 ggtgtgtagc ctagcgcagc gaatagacca gctatccggc ctctggccgg gcatattggg
     1021 caagggcagc agcgccccac aagggcgctg ataaccgcgc ctagtggatt attcttagat
     1081 aatcatggat ggatttttcc aacaccccgc cagcccccgc ccctgctggg tttgcaggtt
     1141 tgggggcgtg acagttattg caggggttcg tgacagttat tgcagggggg cgtgacagtt
     1201 attgcagggg ttcgtgacag ttagtacggg agtgacgggc actggctggc aatgtctagc
     1261 aacggcaggc atttcggctg agggtaaaag aactttccgc taagcgatag actgtatgta
     1321 aacacagtat tgcaaggacg cggaacatgc ctcatgtggc ggccaggacg gccagccggg
     1381 atcgggatac tggtcgttac cagagccacc gacccgagca aacccttctc tatcagatcg
     1441 ttgacgagta ttacccggca ttcgctgcgc ttatggcaga gcagggaaag gaattgccgg
     1501 gctatgtgca acgggaattt gaagaatttc tccaatgcgg gcggctggag catggctttc
     1561 tacgggttcg ctgcgagtct tgccacgccg agcacctggt cgctttcaga aatcaatcta
     1621 aagtatatat gagtaaactt ggtctgacag gcccctgaat tcgcatctag atggtagagc
     1681 cacaaacagc cggtacaagc aacgatctcc aggaccatct gaatcatgcg cggatgacac
     1741 gaactcacga cggcgatcac agacattaac ccacagtaca gacactgcga caacgtggca
     1801 attcgtcgca ataccgtctc actgaactgg ccgataattg cagacgaacg ttatcaaaaa
     1861 gagtattgac ttaaagtcta acctatagga tacttacagc catcgagagg gacacggcga
     1921 ggaattgtga gcggataaca attccataca gaaacagagg agatattaca tatgagtaaa
     1981 ggagaagagc ttttcacagg agttgtccca atcctcgtgg aattagacgg tgatgttaat
     2041 gggcacaagt tctctgtcag tggagagggt gaaggcgacg caacatatgg caagctgacc
     2101 cttaaattta tttgcaccac gggtaaacta cctgttccat ggccaacact ggtcactacg
     2161 ttcgggtatg gggttcagtg ctttgcgcgc tacccagatc acatgaaaca gcacgacttt
     2221 ttcaagagtg caatgcccga aggctatgta caggagagaa ccatcttttt taaggatgac
     2281 ggcaactata agacacgcgc cgaagtgaag ttcgagggtg atacccttgt taatagaatc
     2341 gagttaaagg gtattgactt taaggaagat ggaaatattt taggccacaa actggaatat
     2401 aactataact cccataatgt gtacattatg gccgacaagc aaaagaacgg tatcaaggtt
     2461 aacttcaaga tcagacacaa cattgaggat ggaagcgttc aactagccga ccattaccaa
     2521 caaaacaccc caattggcga tgggcctgtg ctgttaccag acaaccatta cctgtccact
     2581 caatctgccc tttcgaaaga tcccaacgaa aagcgcgacc acatggtcct tcttgagttt
     2641 gtcacggctg ctgggattac acacggcatg gatgaactat acaaataaat ccgtaatcgt
     2701 taatccgcaa ataacgtaaa aacccgcttc ggcgggtttt tttatggggg gagtttaggg
     2761 aaagagcatt tgtcagctgc caatgagacg acggggtcat cacggctcat catgcgccaa
     2821 acaaatgtgt gcaatacacg ctcggatgac tgcatgatga ccgcactgac tggggacagc
     2881 agatccacct aagcctgtga gagaagcaga cacccgacag atcaaggcag ttaactagtg
     2941 cactgcagta caccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
     3001 gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg
     3061 gtgggccttt ctgcgtttat accgagcggc cgcgtgttac aaccaattaa ccaattctga
     3121 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     3181 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     3241 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     3301 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     3361 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
     3421 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     3481 cgcctgagcg aggcgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     3541 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     3601 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     3661 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     3721 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     3781 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     3841 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     3901 cctggagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     3961 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca
     4021 gagattttga gacacaacgt ggctttgttg aataaatcga acttttgctg agttgaagga
     4081 tcagtcatga ccaaaatccc ttaacgtgag tcagcctgcc gccttgggcc gggtgatgtc
     4141 gtacttgccc gccgcgaact cggttaccgt ccagcccagc gcgaccagct ccggcaacgc
     4201 ctcgcgcacc cgctggcggc gcttgcgcat ggtcgaacca ctggcctctg acggccagac
     4261 atagccgcac aaggtatcta tggaagcctt gccggttttg ccggggtcga tccagccaca
     4321 cagccgctgg tgcagcaggc gggcggtttc gctgtccagc gcccgcacct cgtccatgct
     4381 gatgcgcaca tgctggccgc cacccatgac ggcctgcgcg atcaaggggt tcagggccac
     4441 gtacaggcgc ccgtccgcct cgtcgctggc gtactccgac agcagccgaa acccctgccg
     4501 cttgcggcca ttctgggcga tgatggatac cttccaaagg cgctcgatgc agtcctgtat
     4561 gtgcttgagc gccccaccac tatcgacctc tgccccgatt tcctttgcca gcgcccgata
     4621 gctacctttg accaccatgg catcagcggt gacggcctcc cacttgggtt ccaggaacag
     4681 ccggagctgc cgtccgcctt cggtcttggg ttccgggcca agcactaggc cattaggccc
     4741 agccatggcc accagccctt gcaggatgcg cagatcatca gcgcccagcg gctccgggcc
     4801 gctgaactcg atccgcttgc cgtcgccgta gtcatacgtc acgtccagct tgctgcgctt
     4861 gcgctcgccc cgcttgaggg cacggaacag gccgggggcc agacagtgcg ccgggtcgtg
     4921 ccggacgtgg ctgaggctgt gcttgttctt aggcttcacc acggggcacc cccttgctct
     4981 tgcgctgcct ctccagcacg gcgggcttga gcaccccgcc gtcatgccgc ctgaaccacc
     5041 gatcagcgaa cggtgcgcca tagttggcct tgctcacacc gaagcggacg aagaaccggc
     5101 gctggtcgtc gtccacaccc cattcctcgg cctcggcgct ggtcatgctc gacaggtagg
     5161 actgccagcg gatgttatcg accagtaccg agctgccccg gctggcctgc tgctggtcgc
     5221 ctgcgcccat catggccgcg cccttgctgg catggtgcag gaacacgata gagcacccgg
     5281 tatcggcggc gatggcctcc atgcgaccga tgacctgggc catggggccg ctggcgtttt
     5341 cttcctcgat gtggaaccgg cgcagcgtgt ccagcaccat caggcggcgg ccctcggcgg
     5401 cgcgcttgag gccgtcgaac cactccgggg ccatgatgtt gggcaggctg ccgatcagcg
     5461 gctggatcag caggccgtca gccacggctt gccgttcctc ggcgctgagg tgcgccccaa
     5521 gggcgtgcag gcggtgatga atggcggtgg gcgggtcttc ggcgggcagg tagatcaccg
     5581 ggccggtggg cagttcgccc acctccagca gatccggccc gcctgcaatc tgtgcggcca
     5641 gttgcagggc cagcatggat ttaccggcac caccgggcga caccagcgcc ccgaccgtac
     5701 cggccaccat gttgggcaaa acgtagtcca gcggtggcgg cgctgctgcg aacgcctcca
     5761 gaatattgat aggcttatgg gtagccattg attgcctcct ttgcaggcag ttggtggtta
     5821 ggcgctggcg gggtcactac ccccgccctg cgccgctctg agttcttcca ggcactcgcg
     5881 cagcgcctcg tattcgtcgt cggtcagcca gaacttgcgc tgacgcatcc ctttggcctt
     5941 catgcgctcg gcatatcgcg cttggcgtac agcgtcaggg ctggccagca ggtcgccggt
     6001 ctgcttgtcc ttttggtctt tcatatcagt caccgagaaa cttgccgggg ccgaaaggct
     6061 tgtcttcgcg gaacaaggac aaggtgcagc cgtcaaggtt aaggctggcc atatcagcga
     6121 ctgaaaagcg gccagcctcg gccttgtttg acgtataacc aaagccaccg ggcaaccaat
     6181 agcccttgtc acttttgatc aggtagaccg accctgaagc gcttttttcg tattccataa
     6241 aacccccttc tgtgcgtgag tactcatagt ataacaggcg tgagtaccaa cgcaagcact
     6301 acatgctgaa atctggcccg cccctgtcca tgcctcgctg gcggggtgcc ggtgcccgtg
     6361 ccagctcggc ccgcgcaagc tggacgctgg gcagacccat gaccttgctg acggtgcgct
     6421 cgatgtaatc cgcttcgtgg ccgggcttgc gctctgccag cgctgggctg gcctcggcca
     6481 tggccttgcc gatttcctcg gcactgcggc cccggctggc cagcttctgc gcggcgataa
     6541 agtcgcactt gctgaggtca tcaccgaagc gcttgaccag cccggccatc tcgctgcggt
     6601 actcgtccag cgccgtgcgc cggtggcggc taagctgccg ctcgggcagt tcgaggctgg
     6661 ccagcctgcg ggccttctcc tgctgccgct gggcctgctc gatctgctgg ccagcctgct
     6721 gcaccagcgc cgggccagcg gtggcggtct tgcccttgga ttcacgcagc agcacccacg
     6781 gctgataacc ggcgcgggtg gtgtgcttgt ccttgcggtt ggtgaagccc gccaagcggc
     6841 catagtggcg gctgtcggcg ctggccgggt cggcgtcgta ctcgctggcc agcgtccggg
     6901 caatctgccc ccgaagttca ccgcctgcgg cgtcggccac cttgacccat gcctgatagt
     6961 tcttcgggct ggtttccact accagggcag gctcccggcc ctcggctttc atgtcatcca
     7021 ggtcaaactc gctgaggtcg tccaccagca ccagaccatg ccgctcctgc tcggcgggcc
     7081 tgatatacac gtcattgccc tgggcattca tccgcttgag ccatggcgtg ttctggagca
     7141 cttcggcggc tgaccattcc cggttcatca tctggccggt ggtggcgtcc ctgacgccga
     7201 tatcgaagcg ctcacagccc atggccttga gctgtcggcc tatggcctgc aaagtcctgt
     7261 cgttcttcat cgggccacca agcgcagcca gatcgagccg tcctcggttg tcagtggcgt
     7321 caggtcgagc aagagcaacg atgcgatcag cagcaccacc gtaggcatca tggaagccag
     7381 catcacggtt agccatagct tccagtgcca cccccgcgac gcgctccggg cgctctgcgc
     7441 ggcgctgctc acctcggcgg ctacctcccg caactctttg gccagctcca cccatgccgc
     7501 ccctgtctgg cgctgggctt tcagccactc cgccgcctgc gcctcgctgg cctgctgggt
     7561 ctggctcatg acctgccggg cttcgtcggc cagtgtcgcc atgctctggg ccagcggttc
     7621 gatctgctcc gctaactcgt tgatgcctct ggatttcttc actctgtcga ttgcgttcat
     7681 ggtctattgc ctcccggtat tcctgtaagt cgatgatctg ggcgttggcg gtgtcgatgt
     7741 tcagggccac gtctgcccgg tcggtgcgga tgccccggcc ttccatctcc accacgttcg
     7801 gccccaggtg aacaccgggc aggcgctcga tgccctgcgc ctcaagtgtt ctgtggtcaa
     7861 tgcgggcgtc gtggccagcc cgctctaatg cccggttggc atggtcggcc catgcctcgc
     7921 gggtctgctc aagccatgcc ttgggcttga gcgcttcggt cttctgtgcc ccgcccttct
     7981 ccggggtctt gccgttgtac cgcttgaacc actgagcggc gggccgctcg atgccgtcat
     8041 tgatccgctc ggagatcatc aggtggcagt gcgggttctc gccgccaccg gcatggatgg
     8101 ccagcgtata cggcaggc
//
