LOCUS       sequence-218179-        8054 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BTK assembled broad-host-range plasmid encoding GFP driven by CP12b 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8054)
  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 8054)
  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: pBTK619
FEATURES             Location/Qualifiers
     source          1..8054
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            1947..2034
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     rep_origin      2376..2770
                     /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)"
     terminator      4217..4264
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      4453..4524
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4540..4567
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4613..5428)
                     /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(4613..5428)
                     /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     5336..5355
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5420..5439
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(5603..6454)
                     /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(6441..7280)
                     /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(join(7791..8054,1..708))
                     /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 accgaagcgc ttgaccagcc cggccatctc gctgcggtac tcgtccagcg ccgtgcgccg
       61 gtggcggcta agctgccgct cgggcagttc gaggctggcc agcctgcggg ccttctcctg
      121 ctgccgctgg gcctgctcga tctgctggcc agcctgctgc accagcgccg ggccagcggt
      181 ggcggtcttg cccttggatt cacgcagcag cacccacggc tgataaccgg cgcgggtggt
      241 gtgcttgtcc ttgcggttgg tgaagcccgc caagcggcca tagtggcggc tgtcggcgct
      301 ggccgggtcg gcgtcgtact cgctggccag cgtccgggca atctgccccc gaagttcacc
      361 gcctgcggcg tcggccacct tgacccatgc ctgatagttc ttcgggctgg tttccactac
      421 cagggcaggc tcccggccct cggctttcat gtcatccagg tcaaactcgc tgaggtcgtc
      481 caccagcacc agaccatgcc gctcctgctc ggcgggcctg atatacacgt cattgccctg
      541 ggcattcatc cgcttgagcc atggcgtgtt ctggagcact tcggcggctg accattcccg
      601 gttcatcatc tggccggtgg tggcgtccct gacgccgata tcgaagcgct cacagcccat
      661 ggccttgagc tgtcggccta tggcctgcaa agtcctgtcg ttcttcatcg ggccaccaag
      721 cgcagccaga tcgagccgtc ctcggttgtc agtggcgtca ggtcgagcaa gagcaacgat
      781 gcgatcagca gcaccaccgt aggcatcatg gaagccagca tcacggttag ccatagcttc
      841 cagtgccacc cccgcgacgc gctccgggcg ctctgcgcgg cgctgctcac ctcggcggct
      901 acctcccgca actctttggc cagctccacc catgccgccc ctgtctggcg ctgggctttc
      961 agccactccg ccgcctgcgc ctcgctggcc tgctgggtct ggctcatgac ctgccgggct
     1021 tcgtcggcca gtgtcgccat gctctgggcc agcggttcga tctgctccgc taactcgttg
     1081 atgcctctgg atttcttcac tctgtcgatt gcgttcatgg tctattgcct cccggtattc
     1141 ctgtaagtcg atgatctggg cgttggcggt gtcgatgttc agggccacgt ctgcccggtc
     1201 ggtgcggatg ccccggcctt ccatctccac cacgttcggc cccaggtgaa caccgggcag
     1261 gcgctcgatg ccctgcgcct caagtgttct gtggtcaatg cgggcgtcgt ggccagcccg
     1321 ctctaatgcc cggttggcat ggtcggccca tgcctcgcgg gtctgctcaa gccatgcctt
     1381 gggcttgagc gcttcggtct tctgtgcccc gcccttctcc ggggtcttgc cgttgtaccg
     1441 cttgaaccac tgagcggcgg gccgctcgat gccgtcattg atccgctcgg agatcatcag
     1501 gtggcagtgc gggttctcgc cgccaccggc atggatggcc agcgtatacg gcaggcgctc
     1561 ggcaccggtc aggtgctggg cgaactcgga cgccagcgcc ttctgctggt cgagggtcag
     1621 ctcgaccggc agggcaaatt cgacctcctt gaacagccgc ccattggcgc gttcatacag
     1681 gtcggcagca tcccagtagt cggcgggccg ctcgacgaac tccggcatgt gcccggattc
     1741 ggcgtgcaag acttcatcca tgtcgcgggc atacttgcct tcgcgctgga tgtagtcggc
     1801 cttggccctg gccgattggc cgcccgacct gctgccggtt ttcgccgtaa ggtgataaat
     1861 cgccatgctg cctcgctgtt gcttttgctt ttcggctcca tgcaatggcc ctcggagagc
     1921 gcaccgcccg aagggtggcc gttaggccag tttctcgaag agaaaccggt aagtgcgccc
     1981 tcccctacaa agtagggtcg ggattgccgc cgctgtgcct ccatgatagc ctacgagaca
     2041 gcacattaac aatggggtgt caagatggtt aaggggagca acaaggcggc ggatcggctg
     2101 gccaagctcg aagaacaacg agcgcgaatc aatgccgaaa ttcagcgggt gcgggcaagg
     2161 gaacagcagc aagagcgcaa gaacgaaaca aggcgcaagg tgctggtggg ggccatgatt
     2221 ttggccaagg tgaacagcag cgagtggccg gaggatcggc tcatggcggc aatggatgcg
     2281 taccttgaac gcgaccacga ccgcgccttg ttcggtctgc cgccacgcca gaaggatgag
     2341 ccgggctgaa tgatcgaccg agacaggccc tgcggggctg cacacgcgcc cccacccttc
     2401 gggtaggggg aaaggccgct aaagcggcta aaagcgctcc agcgtatttc tgcggggttt
     2461 ggtgtggggt ttagcgggct ttgcccgcct ttccccctgc cgcgcagcgg tggggcggtg
     2521 tgtagcctag cgcagcgaat agaccagcta tccggcctct ggccgggcat attgggcaag
     2581 ggcagcagcg ccccacaagg gcgctgataa ccgcgcctag tggattattc ttagataatc
     2641 atggatggat ttttccaaca ccccgccagc ccccgcccct gctgggtttg caggtttggg
     2701 ggcgtgacag ttattgcagg ggttcgtgac agttattgca ggggggcgtg acagttattg
     2761 caggggttcg tgacagttag tacgggagtg acgggcactg gctggcaatg tctagcaacg
     2821 gcaggcattt cggctgaggg taaaagaact ttccgctaag cgatagactg tatgtaaaca
     2881 cagtattgca aggacgcgga acatgcctca tgtggcggcc aggacggcca gccgggatcg
     2941 ggatactggt cgttaccaga gccaccgacc cgagcaaacc cttctctatc agatcgttga
     3001 cgagtattac ccggcattcg ctgcgcttat ggcagagcag ggaaaggaat tgccgggcta
     3061 tgtgcaacgg gaatttgaag aatttctcca atgcgggcgg ctggagcatg gctttctacg
     3121 ggttcgctgc gagtcttgcc acgccgagca cctggtcgct ttcagaaatc aatctaaagt
     3181 atatatgagt aaacttggtc tgacaggccc ctgaattcgc atctagatgg tagagccaca
     3241 aacagccggt acaagcaacg atctccagga ccatctgaat catgcgcgga tgacacgaac
     3301 tcacgacggc gatcacagac attaacccac agtacagaca ctgcgacaac gtggcaattc
     3361 gtcgcaatac cgtctcactg aactggccga taattgcaga cgaacgcata tacaagttta
     3421 ttcttgacac tagtcggcca aaatgatata atacctgagt actgttatac agaaacagag
     3481 gagatattac atatgagtaa aggagaagag cttttcacag gagttgtccc aatcctcgtg
     3541 gaattagacg gtgatgttaa tgggcacaag ttctctgtca gtggagaggg tgaaggcgac
     3601 gcaacatatg gcaagctgac ccttaaattt atttgcacca cgggtaaact acctgttcca
     3661 tggccaacac tggtcactac gttcgggtat ggggttcagt gctttgcgcg ctacccagat
     3721 cacatgaaac agcacgactt tttcaagagt gcaatgcccg aaggctatgt acaggagaga
     3781 accatctttt ttaaggatga cggcaactat aagacacgcg ccgaagtgaa gttcgagggt
     3841 gatacccttg ttaatagaat cgagttaaag ggtattgact ttaaggaaga tggaaatatt
     3901 ttaggccaca aactggaata taactataac tcccataatg tgtacattat ggccgacaag
     3961 caaaagaacg gtatcaaggt taacttcaag atcagacaca acattgagga tggaagcgtt
     4021 caactagccg accattacca acaaaacacc ccaattggcg atgggcctgt gctgttacca
     4081 gacaaccatt acctgtccac tcaatctgcc ctttcgaaag atcccaacga aaagcgcgac
     4141 cacatggtcc ttcttgagtt tgtcacggct gctgggatta cacacggcat ggatgaacta
     4201 tacaaataaa tcctaactag cataacccct tggggcctct aaacgggtct tgaggggttt
     4261 tttgctggct gagcatgaga cggaaatctg ctcgtcagtg gtgctcacac tgacgaatca
     4321 tgtacagatc ataccgatga ctgcctggcg actcacaact aagcaagaca gccggaacca
     4381 gcgccggcga acaccactgc atatatggca tatcacaaca gtccaactag tgcactgcag
     4441 tacaccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     4501 tgttgtttgt cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct
     4561 ttctgcgttt ataccgagcg gccgcgtgtt acaaccaatt aaccaattct gattagaaaa
     4621 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     4681 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     4741 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     4801 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     4861 gtgagaatgg caaaagctta tgcatttctt tccagacttg ttcaacaggc cagccattac
     4921 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     4981 cgaggcgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     5041 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     5101 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     5161 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     5221 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     5281 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     5341 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctggagc
     5401 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     5461 acagttttat tgttcatgat gatatatttt tatcttgtgc aatgtaacat cagagatttt
     5521 gagacacaac gtggctttgt tgaataaatc gaacttttgc tgagttgaag gatcagtcat
     5581 gaccaaaatc ccttaacgtg agtcagcctg ccgccttggg ccgggtgatg tcgtacttgc
     5641 ccgccgcgaa ctcggttacc gtccagccca gcgcgaccag ctccggcaac gcctcgcgca
     5701 cccgctggcg gcgcttgcgc atggtcgaac cactggcctc tgacggccag acatagccgc
     5761 acaaggtatc tatggaagcc ttgccggttt tgccggggtc gatccagcca cacagccgct
     5821 ggtgcagcag gcgggcggtt tcgctgtcca gcgcccgcac ctcgtccatg ctgatgcgca
     5881 catgctggcc gccacccatg acggcctgcg cgatcaaggg gttcagggcc acgtacaggc
     5941 gcccgtccgc ctcgtcgctg gcgtactccg acagcagccg aaacccctgc cgcttgcggc
     6001 cattctgggc gatgatggat accttccaaa ggcgctcgat gcagtcctgt atgtgcttga
     6061 gcgccccacc actatcgacc tctgccccga tttcctttgc cagcgcccga tagctacctt
     6121 tgaccaccat ggcatcagcg gtgacggcct cccacttggg ttccaggaac agccggagct
     6181 gccgtccgcc ttcggtcttg ggttccgggc caagcactag gccattaggc ccagccatgg
     6241 ccaccagccc ttgcaggatg cgcagatcat cagcgcccag cggctccggg ccgctgaact
     6301 cgatccgctt gccgtcgccg tagtcatacg tcacgtccag cttgctgcgc ttgcgctcgc
     6361 cccgcttgag ggcacggaac aggccggggg ccagacagtg cgccgggtcg tgccggacgt
     6421 ggctgaggct gtgcttgttc ttaggcttca ccacggggca cccccttgct cttgcgctgc
     6481 ctctccagca cggcgggctt gagcaccccg ccgtcatgcc gcctgaacca ccgatcagcg
     6541 aacggtgcgc catagttggc cttgctcaca ccgaagcgga cgaagaaccg gcgctggtcg
     6601 tcgtccacac cccattcctc ggcctcggcg ctggtcatgc tcgacaggta ggactgccag
     6661 cggatgttat cgaccagtac cgagctgccc cggctggcct gctgctggtc gcctgcgccc
     6721 atcatggccg cgcccttgct ggcatggtgc aggaacacga tagagcaccc ggtatcggcg
     6781 gcgatggcct ccatgcgacc gatgacctgg gccatggggc cgctggcgtt ttcttcctcg
     6841 atgtggaacc ggcgcagcgt gtccagcacc atcaggcggc ggccctcggc ggcgcgcttg
     6901 aggccgtcga accactccgg ggccatgatg ttgggcaggc tgccgatcag cggctggatc
     6961 agcaggccgt cagccacggc ttgccgttcc tcggcgctga ggtgcgcccc aagggcgtgc
     7021 aggcggtgat gaatggcggt gggcgggtct tcggcgggca ggtagatcac cgggccggtg
     7081 ggcagttcgc ccacctccag cagatccggc ccgcctgcaa tctgtgcggc cagttgcagg
     7141 gccagcatgg atttaccggc accaccgggc gacaccagcg ccccgaccgt accggccacc
     7201 atgttgggca aaacgtagtc cagcggtggc ggcgctgctg cgaacgcctc cagaatattg
     7261 ataggcttat gggtagccat tgattgcctc ctttgcaggc agttggtggt taggcgctgg
     7321 cggggtcact acccccgccc tgcgccgctc tgagttcttc caggcactcg cgcagcgcct
     7381 cgtattcgtc gtcggtcagc cagaacttgc gctgacgcat ccctttggcc ttcatgcgct
     7441 cggcatatcg cgcttggcgt acagcgtcag ggctggccag caggtcgccg gtctgcttgt
     7501 ccttttggtc tttcatatca gtcaccgaga aacttgccgg ggccgaaagg cttgtcttcg
     7561 cggaacaagg acaaggtgca gccgtcaagg ttaaggctgg ccatatcagc gactgaaaag
     7621 cggccagcct cggccttgtt tgacgtataa ccaaagccac cgggcaacca atagcccttg
     7681 tcacttttga tcaggtagac cgaccctgaa gcgctttttt cgtattccat aaaaccccct
     7741 tctgtgcgtg agtactcata gtataacagg cgtgagtacc aacgcaagca ctacatgctg
     7801 aaatctggcc cgcccctgtc catgcctcgc tggcggggtg ccggtgcccg tgccagctcg
     7861 gcccgcgcaa gctggacgct gggcagaccc atgaccttgc tgacggtgcg ctcgatgtaa
     7921 tccgcttcgt ggccgggctt gcgctctgcc agcgctgggc tggcctcggc catggccttg
     7981 ccgatttcct cggcactgcg gccccggctg gccagcttct gcgcggcgat aaagtcgcac
     8041 ttgctgaggt catc
//
