LOCUS       sequence-208065-        7536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BTK Assembled plasmid- ready for Stage 2 assembly (ConLS' and ConRE'
            connectors with kanamycin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7536)
  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 7536)
  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: pBTK527
FEATURES             Location/Qualifiers
     source          1..7536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            1855..1942
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     rep_origin      2284..2678
                     /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      3843..3914
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3930..3957
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(4003..4818)
                     /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(4003..4818)
                     /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     4810..4829
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             complement(4993..5844)
                     /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
                     TAECHVVKGSYRALAKEIGAEVDSGGALKHIQDCIERLWKVSIIAQNGRKRQGFRLLSE
                     YASDEADGRLYVALNPLIAQAVMGGGQHVRISMDEVRALDSETARLLHQRLCGWIDPGK
                     TGKASIDTLCGYVWPSEASGSTMRKRRKRVREALPELVALGWTVTEFAAGKYDITRPKA
                     AG"
     CDS             complement(5831..6670)
                     /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(7181..7536,1..616))
                     /codon_start=1
                     /gene="repB"
                     /product="replication protein B of the broad-host-range 
                     plasmid RSF1010 (Scholz et al., 1989)"
                     /label=RSF1010 RepB
                     /translation="MKNDRTLQAIGRQLKAMGCERFDIGVRDAPTGQMMNREWSAAEVL
                     QNTPWLKRMNAQGNDVYIRPAEQERHGLVLVDDLSEFDLDDMKAEGREPALVVETSPKN
                     YQAWVKVADAAGGELRGQIARTLASEYDADPASADSRHYGRLAGFTNRKDKHTTRAGYQ
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELPERQLSRHRRTALD
                     EYRSEMAGLVKRFGHDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
ORIGIN
        1 ggctggccag cctgcgggcc ttctcctgct gccgctgggc ctgctcgatc tgctggccag
       61 cctgctgcac cagcgccggg ccagcggtgg cggtcttgcc cttggattca cgcagcagca
      121 cccacggctg ataaccggcg cgggtggtgt gcttgtcctt gcggttggtg aagcccgcca
      181 agcggccata gtggcggctg tcggcgctgg ccgggtcggc gtcgtactcg ctggccagcg
      241 tccgggcaat ctgcccccga agttcaccgc ctgcggcgtc ggccaccttg acccatgcct
      301 gatagttctt cgggctggtt tccactacca gggcaggctc ccggccctcg gctttcatgt
      361 catccaggtc aaactcgctg aggtcgtcca ccagcaccag accatgccgc tcctgctcgg
      421 cgggcctgat atacacgtca ttgccctggg cattcatccg cttgagccat ggcgtgttct
      481 ggagcacttc ggcggctgac cattcccggt tcatcatctg gccggtgggt gcgtccctga
      541 cgccgatatc gaagcgctca cagcccatgg ccttgagctg tcggcctatg gcctgcaaag
      601 tcctgtcgtt cttcatcggg ccaccaagcg cagccagatc gagccgtcct cggttgtcag
      661 tggcgtcagg tcgagcaaga gcaacgatgc gatcagcagc accaccgtag gcatcatgga
      721 agccagcatc acggttagcc atagcttcca gtgccacccc cgcgacgcgc tccgggcgct
      781 ctgcgcggcg ctgctcacct cggcggctac ctcccgcaac tctttggcca gctccaccca
      841 tgccgcccct gtctggcgct gggctttcag ccactccgcc gcctgcgcct cgctggcctg
      901 cttggtctgg ctcatgacct gccgggcttc gtcggccagt gtcgccatgc tctgggccag
      961 cggttcgatc tgctccgcta actcgttgat gcctctggat ttcttcactc tgtcgattgc
     1021 gttcatggtc tattgcctcc cggtattcct gtaagtcgat gatctgggcg ttggcggtgt
     1081 cgatgttcag ggccacgtct gcccggtcgg tgcggatgcc ccggccttcc atctccacca
     1141 cgttcggccc caggtgaaca ccgggcaggc gctcgatgcc ctgcgcctca agtgttctgt
     1201 ggtcaatgcg ggcgtcgtgg ccagcccgct ctaatgcccg gttggcatgg tcggcccatg
     1261 cctcgcgggt ctgctcaagc catgccttgg gcttgagcgc ttcggtcttc tgtgccccgc
     1321 ccttctccgg ggtcttgccg ttgtaccgct tgaaccactg agcggcgggc cgctcgatgc
     1381 cgtcattgat ccgctcggag atcatcaggt ggcagtgcgg gttctcgccg ccaccggcat
     1441 ggatggccag cgtatacggc aggcgctcgg caccggtcag gtgctgggcg aactcggacg
     1501 ccagcgcctt ctgctggtcg agggtcagct cgaccggcag ggcaaattcg acctccttga
     1561 acagccgccc attggcgcgt tcatacaggt cggcagcatc ccagtagtcg gcgggccgct
     1621 cgacgaactc cggcatgtgc ccggattcgg cgtgcaagac ttcatccatg tcgcgggcat
     1681 acttgccttc gcgctggatg tagtcggcct tggccctggc cgattggccg cccgacctgc
     1741 tgccggtttt cgccgtaagg tgataaatcg ccatgctgcc tcgctgttgc ttttgctttt
     1801 cggctccatg caatggccct cggagagcgc accgcccgaa gggtggccgt taggccagtt
     1861 tctcgaagag aaaccggtaa gtgcgccctc ccctacaaag tagggtcggg attgccgccg
     1921 ctgtgcctcc atgatagcct acgagacagc acattaacaa tggggtgtca agatggttaa
     1981 ggggagcaac aaggcggcgg atcggctggc caagctcgaa gaacaacgag cgcgaatcaa
     2041 tgccgaaatt cagcgggagc gggcaaggga acagcagcaa gagcgcaaga acgaaacaag
     2101 gcgcaaggtg ctggtggggg ccatgatttt ggccaaggtg aacagcagcg agtggccgga
     2161 ggatcggctc atggcggcaa tggatgcgta ccttgaacgc gaccacgacc gcgccttgtt
     2221 cggtctgccg ccacgccaga aggatgagcc gggctgaatg atcgaccgag acaggccctg
     2281 cggggctgca cacgcgcccc cacccttcgg gtagggggaa aggccgctaa agcggctaaa
     2341 agcgctccag cgtatttctg cggggtttgg tgtggggttt agcgggcttt gcccgccttt
     2401 ccccctgccg cgcagcggtg gggcggtgtg tagcctagcg cagcgaatag accagctatc
     2461 cggcctctgg ccgggcatat tgggcaaggg cagcagcgcc ccacaagggc gctgataacc
     2521 gcgcctagtg gattattctt agataatcat ggatggattt ttccaacacc ccgccagccc
     2581 ccgcccctgc tgggtttgca ggtttggggg cgtgacagtt attgcagggg ttcgtgacag
     2641 ttattgcagg ggggcgtgac agttattgca ggggttcgtg acagttagta cgggagtgac
     2701 gggcactggc tggcaatgtc tagcaacggc aggcatttcg gctgagggta aaagaacttt
     2761 ccgctaagcg atagactgta tgtaaacaca gtattgcaag gacgcggaac atgcctcatg
     2821 tggcggccag gacggccagc cgggatcggg atactggtcg ttaccagagc caccgacccg
     2881 agcaaaccct tctctatcag atcgttgacg agtattaccc ggcattcgct gcgcttatgg
     2941 cagagcaggg aaaggaattg ccgggctatg tgcaacggga atttgaagaa tttctccaat
     3001 gcgggcggct ggagcatggc tttctacggg ttcgctgcga gtcttgccac gccgagcacc
     3061 tggtcgcttt cagaaatcaa tctaaagtat atatgagtaa acttggtctg acaggcccct
     3121 gaattcgcat ctagactgat gagacgtggt agagccacaa acagccggta caagcaacga
     3181 tctccaggac catctgaatc atgcgcggat gacacgaact cacgacggcg atcacagaca
     3241 ttaacccaca gtacagacac tgcgacaacg tggcaattcg tcgcaataca acgtaacggg
     3301 cttcccatac aatcgattag agattgtcat cgcacctgat tgcccgacat tatcgcgagt
     3361 tataacccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcgtgagcct
     3421 atgtaacggg cttcccatac aatcgattag agattgtcat cgcacctgat tgcccgacat
     3481 tatcgcgagt tataacccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     3541 gcgtgagcca tccgtaatcg ttaatccgca aataacgtaa aaacccgctt cggcgggttt
     3601 ttttatgggg ggagtttagg gaaagagcat ttgtcagctg gaaatctgct cgtcagtggt
     3661 gctcacactg acgaatcatg tacagatcat accgatgact gcctggcgac tcacaactaa
     3721 gcaagacagc cggaaccagc gccggcgaac accactgcat atatggcata tcacaacagt
     3781 ccacgtctca agcagttaca gagatgttac gaaccactag tgcactgcag tacaccaggc
     3841 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     3901 cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct ttctgcgttt
     3961 ataccgagcg gccgcgtgtt acaaccaatt aaccaattct gattagaaaa actcatcgag
     4021 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     4081 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     4141 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     4201 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     4261 caaaagctta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     4321 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgaggcgaaa
     4381 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     4441 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     4501 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     4561 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     4621 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     4681 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     4741 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctggagc aagacgtttc
     4801 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     4861 tgttcatgat gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacaac
     4921 gtggctttgt tgaataaatc gaacttttgc tgagttgaag gatcagtcat gaccaaaatc
     4981 ccttaacgtg agtcagcctg ccgccttggg ccgggtgatg tcgtacttgc ccgccgcgaa
     5041 ctcggttacc gtccagccca gcgcgaccag ctccggcaac gcctcgcgca cccgcttgcg
     5101 gcgcttgcgc atggtcgaac cactggcctc tgacggccag acatagccgc acaaggtatc
     5161 tatggaagcc ttgccggttt tgccggggtc gatccagcca cacagccgct ggtgcagcag
     5221 gcgggcggtt tcgctgtcca gcgcccgcac ctcgtccatg ctgatgcgca catgctggcc
     5281 gccacccatg acggcctgcg cgatcaaggg gttcagggcc acgtacaggc gcccgtccgc
     5341 ctcgtcgctg gcgtactccg acagcagccg aaacccctgc cgcttgcggc cattctgggc
     5401 gatgatggat accttccaaa ggcgctcgat gcagtcctgt atgtgcttga gcgccccacc
     5461 actatcgacc tctgccccga tttcctttgc cagcgcccga tagctacctt tgaccacatg
     5521 gcattcagcg gtgacggcct cccacttggg ttccaggaac agccggagct gccgtccgcc
     5581 ttcggtcttg ggttccgggc caagcactag gccattaggc ccagccatgg ccaccagccc
     5641 ttgcaggatg cgcagatcat cagcgcccag cggctccggg ccgctgaact cgatccgctt
     5701 gccgtcgccg tagtcatacg tcacgtccag cttgctgcgc ttgcgctcgc cccgcttgag
     5761 ggcacggaac aggccggggg ccagacagtg cgccgggtcg tgccggacgt ggctgaggct
     5821 gtgcttgttc ttaggcttca ccacggggca cccccttgct cttgcgctgc ctctccagca
     5881 cggcgggctt gagcaccccg ccgtcatgcc gcctgaacca ccgatcagcg aacggtgcgc
     5941 catagttggc cttgctcaca ccgaagcgga cgaagaaccg gcgctggtcg tcgtccacac
     6001 cccattcctc ggcctcggcg ctggtcatgc tcgacaggta ggactgccag cggatgttat
     6061 cgaccagtac cgagctgccc cggctggcct gctgctggtc gcctgcgccc atcatggccg
     6121 cgcccttgct ggcatggtgc aggaacacga tagagcaccc ggtatcggcg gcgatggcct
     6181 ccatgcgacc gatgacctgg gccatggggc cgctggcgtt ttcttcctcg atgtggaacc
     6241 ggcgcagcgt gtccagcacc atcaggcggc ggccctcggc ggcgcgcttg aggccgtcga
     6301 accactccgg ggccatgatg ttgggcaggc tgccgatcag cggctggatc agcaggccgt
     6361 cagccacggc ttgccgttcc tcggcgctga ggtgcgcccc aagggcgtgc aggcggtgat
     6421 gaatggcggt gggcgggtct tcggcgggca ggtagatcac cgggccggtg ggcagttcgc
     6481 ccacctccag cagatccggc ccgcctgcaa tctgtgcggc cagttgcagg gccagcatgg
     6541 atttaccggc accaccgggc gacaccagcg ccccgaccgt accggccacc atgttgggca
     6601 aaacgtagtc cagcggtggc ggcgctgctg cgaacgcctc cagaatattg ataggcttat
     6661 gggtagccat tgattgcctc ctttgcaggc agttggtggt taggcgctgg cggggtcact
     6721 acccccgccc tgcgccgctc tgagttcttc caggcactcg cgcagcgcct cgtattcgtc
     6781 gtcggtcagc cagaacttgc gctgacgcat ccctttggcc ttcatgcgct cggcatatcg
     6841 cgcttggcgt acagcgtcag ggctggccag caggtcgccg gtctgcttgt ccttttggtc
     6901 tttcatatca gtcaccgaga aacttgccgg ggccgaaagg cttgtcttcg cggaacaagg
     6961 acaaggtgca gccgtcaagg ttaaggctgg ccatatcagc gactgaaaag cggccagcct
     7021 cggccttgtt tgacgtataa ccaaagccac cgggcaacca atagcccttg tcacttttga
     7081 tcaggtagac cgaccctgaa gcgctttttt cgtattccat aaaaccccct tctgtgcgtg
     7141 agtactcata gtataacagg cgtgagtacc aacgcaagca ctacatgctg aaatctggcc
     7201 cgcccctgtc catgcctcgc tggcggggtg ccggtgcccg tgccagctcg gcccgcgcaa
     7261 gctggacgct gggcagaccc atgaccttgc tgacggtgcg ctcgatgtaa tccgcttcgt
     7321 ggccgggctt gcgctctgcc agcgctgggc tggcctcggc catggccttg ccgatttcct
     7381 cggcactgcg gccccggctg gccagcttct gcgcggcgat aaagtcgcac ttgctgaggt
     7441 catgaccgaa gcgcttgacc agcccggcca tctcgctgcg gtactcgtcc agcgccgtgc
     7501 gccggtggcg gctaagctgc cgctcgggca gttcga
//
