LOCUS       sequence-233533-        8054 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BTK assembled broad-host-range plasmid encoding GFP driven by CP32 
            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: pBTK620
FEATURES             Location/Qualifiers
     source          1..8054
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            1660..1747
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     rep_origin      2089..2483
                     /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      3930..3977
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     terminator      4166..4237
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4253..4280
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4326..5141)
                     /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(4326..5141)
                     /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     5049..5068
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5133..5152
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(5316..6167)
                     /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(6154..6993)
                     /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(7504..8054,1..421))
                     /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 ggctgtcggc gctggccggg tcggcgtcgt actcgctggc cagcgtccgg gcaatctgcc
       61 cccgaagttc accgcctgcg gcgtcggcca ccttgaccca tgcctgatag ttcttcgggc
      121 tggtttccac taccagggca ggctcccggc cctcggcttt catgtcatcc aggtcaaact
      181 cgctgaggtc gtccaccagc accagaccat gccgctcctg ctcggcgggc ctgatataca
      241 cgtcattgcc ctgggcattc atccgcttga gccatggcgt gttctggagc acttcggcgg
      301 ctgaccattc ccggttcatc atctggccgg tggtggcgtc cctgacgccg atatcgaagc
      361 gctcacagcc catggccttg agctgtcggc ctatggcctg caaagtcctg tcgttcttca
      421 tcgggccacc aagcgcagcc agatcgagcc gtcctcggtt gtcagtggcg tcaggtcgag
      481 caagagcaac gatgcgatca gcagcaccac cgtaggcatc atggaagcca gcatcacggt
      541 tagccatagc ttccagtgcc acccccgcga cgcgctccgg gcgctctgcg cggcgctgct
      601 cacctcggcg gctacctccc gcaactcttt ggccagctcc acccatgccg cccctgtctg
      661 gcgctgggct ttcagccact ccgccgcctg cgcctcgctg gcctgctggg tctggctcat
      721 gacctgccgg gcttcgtcgg ccagtgtcgc catgctctgg gccagcggtt cgatctgctc
      781 cgctaactcg ttgatgcctc tggatttctt cactctgtcg attgcgttca tggtctattg
      841 cctcccggta ttcctgtaag tcgatgatct gggcgttggc ggtgtcgatg ttcagggcca
      901 cgtctgcccg gtcggtgcgg atgccccggc cttccatctc caccacgttc ggccccaggt
      961 gaacaccggg caggcgctcg atgccctgcg cctcaagtgt tctgtggtca atgcgggcgt
     1021 cgtggccagc ccgctctaat gcccggttgg catggtcggc ccatgcctcg cgggtctgct
     1081 caagccatgc cttgggcttg agcgcttcgg tcttctgtgc cccgcccttc tccggggtct
     1141 tgccgttgta ccgcttgaac cactgagcgg cgggccgctc gatgccgtca ttgatccgct
     1201 cggagatcat caggtggcag tgcgggttct cgccgccacc ggcatggatg gccagcgtat
     1261 acggcaggcg ctcggcaccg gtcaggtgct gggcgaactc ggacgccagc gccttctgct
     1321 ggtcgagggt cagctcgacc ggcagggcaa attcgacctc cttgaacagc cgcccattgg
     1381 cgcgttcata caggtcggca gcatcccagt agtcggcggg ccgctcgacg aactccggca
     1441 tgtgcccgga ttcggcgtgc aagacttcat ccatgtcgcg ggcatacttg ccttcgcgct
     1501 ggatgtagtc ggccttggcc ctggccgatt ggccgcccga cctgctgccg gttttcgccg
     1561 taaggtgata aatcgccatg ctgcctcgct gttgcttttg cttttcggct ccatgcaatg
     1621 gccctcggag agcgcaccgc ccgaagggtg gccgttaggc cagtttctcg aagagaaacc
     1681 ggtaagtgcg ccctccccta caaagtaggg tcgggattgc cgccgctgtg cctccatgat
     1741 agcctacgag acagcacatt aacaatgggg tgtcaagatg gttaagggga gcaacaaggc
     1801 ggcggatcgg ctggccaagc tcgaagaaca acgagcgcga atcaatgccg aaattcagcg
     1861 ggtgcgggca agggaacagc agcaagagcg caagaacgaa acaaggcgca aggtgctggt
     1921 gggggccatg attttggcca aggtgaacag cagcgagtgg ccggaggatc ggctcatggc
     1981 ggcaatggat gcgtaccttg aacgcgacca cgaccgcgcc ttgttcggtc tgccgccacg
     2041 ccagaaggat gagccgggct gaatgatcga ccgagacagg ccctgcgggg ctgcacacgc
     2101 gcccccaccc ttcgggtagg gggaaaggcc gctaaagcgg ctaaaagcgc tccagcgtat
     2161 ttctgcgggg tttggtgtgg ggtttagcgg gctttgcccg cctttccccc tgccgcgcag
     2221 cggtggggcg gtgtgtagcc tagcgcagcg aatagaccag ctatccggcc tctggccggg
     2281 catattgggc aagggcagca gcgccccaca agggcgctga taaccgcgcc tagtggatta
     2341 ttcttagata atcatggatg gatttttcca acaccccgcc agcccccgcc cctgctgggt
     2401 ttgcaggttt gggggcgtga cagttattgc aggggttcgt gacagttatt gcaggggggc
     2461 gtgacagtta ttgcaggggt tcgtgacagt tagtacggga gtgacgggca ctggctggca
     2521 atgtctagca acggcaggca tttcggctga gggtaaaaga actttccgct aagcgataga
     2581 ctgtatgtaa acacagtatt gcaaggacgc ggaacatgcc tcatgtggcg gccaggacgg
     2641 ccagccggga tcgggatact ggtcgttacc agagccaccg acccgagcaa acccttctct
     2701 atcagatcgt tgacgagtat tacccggcat tcgctgcgct tatggcagag cagggaaagg
     2761 aattgccggg ctatgtgcaa cgggaatttg aagaatttct ccaatgcggg cggctggagc
     2821 atggctttct acgggttcgc tgcgagtctt gccacgccga gcacctggtc gctttcagaa
     2881 atcaatctaa agtatatatg agtaaacttg gtctgacagg cccctgaatt cgcatctaga
     2941 tggtagagcc acaaacagcc ggtacaagca acgatctcca ggaccatctg aatcatgcgc
     3001 ggatgacacg aactcacgac ggcgatcaca gacattaacc cacagtacag acactgcgac
     3061 aacgtggcaa ttcgtcgcaa taccgtctca ctgaactggc cgataattgc agacgaacgc
     3121 atacgggagt ttattcttga catattgccg gtgtgttggt ataataactt agtactgtta
     3181 tacagaaaca gaggagatat tacatatgag taaaggagaa gagcttttca caggagttgt
     3241 cccaatcctc gtggaattag acggtgatgt taatgggcac aagttctctg tcagtggaga
     3301 gggtgaaggc gacgcaacat atggcaagct gacccttaaa tttatttgca ccacgggtaa
     3361 actacctgtt ccatggccaa cactggtcac tacgttcggg tatggggttc agtgctttgc
     3421 gcgctaccca gatcacatga aacagcacga ctttttcaag agtgcaatgc ccgaaggcta
     3481 tgtacaggag agaaccatct tttttaagga tgacggcaac tataagacac gcgccgaagt
     3541 gaagttcgag ggtgataccc ttgttaatag aatcgagtta aagggtattg actttaagga
     3601 agatggaaat attttaggcc acaaactgga atataactat aactcccata atgtgtacat
     3661 tatggccgac aagcaaaaga acggtatcaa ggttaacttc aagatcagac acaacattga
     3721 ggatggaagc gttcaactag ccgaccatta ccaacaaaac accccaattg gcgatgggcc
     3781 tgtgctgtta ccagacaacc attacctgtc cactcaatct gccctttcga aagatcccaa
     3841 cgaaaagcgc gaccacatgg tccttcttga gtttgtcacg gctgctggga ttacacacgg
     3901 catggatgaa ctatacaaat aaatcctaac tagcataacc ccttggggcc tctaaacggg
     3961 tcttgagggg ttttttgctg gctgagcatg agacggaaat ctgctcgtca gtggtgctca
     4021 cactgacgaa tcatgtacag atcataccga tgactgcctg gcgactcaca actaagcaag
     4081 acagccggaa ccagcgccgg cgaacaccac tgcatatatg gcatatcaca acagtccaac
     4141 tagtgcactg cagtacacca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     4201 ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc
     4261 ttcgggtggg cctttctgcg tttataccga gcggccgcgt gttacaacca attaaccaat
     4321 tctgattaga aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta
     4381 tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag
     4441 ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata
     4501 caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg
     4561 acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac ttgttcaaca
     4621 ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt
     4681 gattgcgcct gagcgaggcg aaatacgcga tcgctgttaa aaggacaatt acaaacagga
     4741 atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca
     4801 ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat
     4861 gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc
     4921 cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc
     4981 agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc
     5041 ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat
     5101 cgcggcctgg agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg
     5161 tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa
     5221 catcagagat tttgagacac aacgtggctt tgttgaataa atcgaacttt tgctgagttg
     5281 aaggatcagt catgaccaaa atcccttaac gtgagtcagc ctgccgcctt gggccgggtg
     5341 atgtcgtact tgcccgccgc gaactcggtt accgtccagc ccagcgcgac cagctccggc
     5401 aacgcctcgc gcacccgctg gcggcgcttg cgcatggtcg aaccactggc ctctgacggc
     5461 cagacatagc cgcacaaggt atctatggaa gccttgccgg ttttgccggg gtcgatccag
     5521 ccacacagcc gctggtgcag caggcgggcg gtttcgctgt ccagcgcccg cacctcgtcc
     5581 atgctgatgc gcacatgctg gccgccaccc atgacggcct gcgcgatcaa ggggttcagg
     5641 gccacgtaca ggcgcccgtc cgcctcgtcg ctggcgtact ccgacagcag ccgaaacccc
     5701 tgccgcttgc ggccattctg ggcgatgatg gataccttcc aaaggcgctc gatgcagtcc
     5761 tgtatgtgct tgagcgcccc accactatcg acctctgccc cgatttcctt tgccagcgcc
     5821 cgatagctac ctttgaccac catggcatca gcggtgacgg cctcccactt gggttccagg
     5881 aacagccgga gctgccgtcc gccttcggtc ttgggttccg ggccaagcac taggccatta
     5941 ggcccagcca tggccaccag cccttgcagg atgcgcagat catcagcgcc cagcggctcc
     6001 gggccgctga actcgatccg cttgccgtcg ccgtagtcat acgtcacgtc cagcttgctg
     6061 cgcttgcgct cgccccgctt gagggcacgg aacaggccgg gggccagaca gtgcgccggg
     6121 tcgtgccgga cgtggctgag gctgtgcttg ttcttaggct tcaccacggg gcaccccctt
     6181 gctcttgcgc tgcctctcca gcacggcggg cttgagcacc ccgccgtcat gccgcctgaa
     6241 ccaccgatca gcgaacggtg cgccatagtt ggccttgctc acaccgaagc ggacgaagaa
     6301 ccggcgctgg tcgtcgtcca caccccattc ctcggcctcg gcgctggtca tgctcgacag
     6361 gtaggactgc cagcggatgt tatcgaccag taccgagctg ccccggctgg cctgctgctg
     6421 gtcgcctgcg cccatcatgg ccgcgccctt gctggcatgg tgcaggaaca cgatagagca
     6481 cccggtatcg gcggcgatgg cctccatgcg accgatgacc tgggccatgg ggccgctggc
     6541 gttttcttcc tcgatgtgga accggcgcag cgtgtccagc accatcaggc ggcggccctc
     6601 ggcggcgcgc ttgaggccgt cgaaccactc cggggccatg atgttgggca ggctgccgat
     6661 cagcggctgg atcagcaggc cgtcagccac ggcttgccgt tcctcggcgc tgaggtgcgc
     6721 cccaagggcg tgcaggcggt gatgaatggc ggtgggcggg tcttcggcgg gcaggtagat
     6781 caccgggccg gtgggcagtt cgcccacctc cagcagatcc ggcccgcctg caatctgtgc
     6841 ggccagttgc agggccagca tggatttacc ggcaccaccg ggcgacacca gcgccccgac
     6901 cgtaccggcc accatgttgg gcaaaacgta gtccagcggt ggcggcgctg ctgcgaacgc
     6961 ctccagaata ttgataggct tatgggtagc cattgattgc ctcctttgca ggcagttggt
     7021 ggttaggcgc tggcggggtc actacccccg ccctgcgccg ctctgagttc ttccaggcac
     7081 tcgcgcagcg cctcgtattc gtcgtcggtc agccagaact tgcgctgacg catccctttg
     7141 gccttcatgc gctcggcata tcgcgcttgg cgtacagcgt cagggctggc cagcaggtcg
     7201 ccggtctgct tgtccttttg gtctttcata tcagtcaccg agaaacttgc cggggccgaa
     7261 aggcttgtct tcgcggaaca aggacaaggt gcagccgtca aggttaaggc tggccatatc
     7321 agcgactgaa aagcggccag cctcggcctt gtttgacgta taaccaaagc caccgggcaa
     7381 ccaatagccc ttgtcacttt tgatcaggta gaccgaccct gaagcgcttt tttcgtattc
     7441 cataaaaccc ccttctgtgc gtgagtactc atagtataac aggcgtgagt accaacgcaa
     7501 gcactacatg ctgaaatctg gcccgcccct gtccatgcct cgctggcggg gtgccggtgc
     7561 ccgtgccagc tcggcccgcg caagctggac gctgggcaga cccatgacct tgctgacggt
     7621 gcgctcgatg taatccgctt cgtggccggg cttgcgctct gccagcgctg ggctggcctc
     7681 ggccatggcc ttgccgattt cctcggcact gcggccccgg ctggccagct tctgcgcggc
     7741 gataaagtcg cacttgctga ggtcatcacc gaagcgcttg accagcccgg ccatctcgct
     7801 gcggtactcg tccagcgccg tgcgccggtg gcggctaagc tgccgctcgg gcagttcgag
     7861 gctggccagc ctgcgggcct tctcctgctg ccgctgggcc tgctcgatct gctggccagc
     7921 ctgctgcacc agcgccgggc cagcggtggc ggtcttgccc ttggattcac gcagcagcac
     7981 ccacggctga taaccggcgc gggtggtgtg cttgtccttg cggttggtga agcccgccaa
     8041 gcggccatag tggc
//
