LOCUS       sequence-217970-        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"
     rep_origin      865..1259
                     /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            1601..1688
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     CDS             2927..3898
                     /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"
     CDS             4409..5248
                     /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             5235..6086
                     /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"
     primer_bind     complement(6250..6269)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6261..7076
                     /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             6261..7076
                     /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"
     terminator      complement(7122..7149)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(7165..7236)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggtta taactcgcga
       61 taatgtcggg caatcaggtg cgatgacaat ctctaatcga ttgtatggga agcccgttac
      121 ataggctcac gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggtta
      181 taactcgcga taatgtcggg caatcaggtg cgatgacaat ctctaatcga ttgtatggga
      241 agcccgttac gttgtattgc gacgaattgc cacgttgtcg cagtgtctgt actgtgggtt
      301 aatgtctgtg atcgccgtcg tgagttcgtg tcatccgcgc atgattcaga tggtcctgga
      361 gatcgttgct tgtaccggct gtttgtggct ctaccacgtc tcatcagtct agatgcgaat
      421 tcaggggcct gtcagaccaa gtttactcat atatacttta gattgatttc tgaaagcgac
      481 caggtgctcg gcgtggcaag actcgcagcg aacccgtaga aagccatgct ccagccgccc
      541 gcattggaga aattcttcaa attcccgttg cacatagccc ggcaattcct ttccctgctc
      601 tgccataagc gcagcgaatg ccgggtaata ctcgtcaacg atctgataga gaagggtttg
      661 ctcgggtcgg tggctctggt aacgaccagt atcccgatcc cggctggccg tcctggccgc
      721 cacatgaggc atgttccgcg tccttgcaat actgtgttta catacagtct atcgcttagc
      781 ggaaagttct tttaccctca gccgaaatgc ctgccgttgc tagacattgc cagccagtgc
      841 ccgtcactcc cgtactaact gtcacgaacc cctgcaataa ctgtcacgcc cccctgcaat
      901 aactgtcacg aacccctgca ataactgtca cgcccccaaa cctgcaaacc cagcaggggc
      961 gggggctggc ggggtgttgg aaaaatccat ccatgattat ctaagaataa tccactaggc
     1021 gcggttatca gcgcccttgt ggggcgctgc tgcccttgcc caatatgccc ggccagaggc
     1081 cggatagctg gtctattcgc tgcgctaggc tacacaccgc cccaccgctg cgcggcaggg
     1141 ggaaaggcgg gcaaagcccg ctaaacccca caccaaaccc cgcagaaata cgctggagcg
     1201 cttttagccg ctttagcggc ctttccccct acccgaaggg tgggggcgcg tgtgcagccc
     1261 cgcagggcct gtctcggtcg atcattcagc ccggctcatc cttctggcgt ggcggcagac
     1321 cgaacaaggc gcggtcgtgg tcgcgttcaa ggtacgcatc cattgccgcc atgagccgat
     1381 cctccggcca ctcgctgctg ttcaccttgg ccaaaatcat ggcccccacc agcaccttgc
     1441 gccttgtttc gttcttgcgc tcttgctgct gttcccttgc ccgcacccgc tgaatttcgg
     1501 cattgattcg cgctcgttgt tcttcgagct tggccagccg atccgccgcc ttgttgctcc
     1561 ccttaaccat cttgacaccc cattgttaat gtgctgtctc gtaggctatc atggaggcac
     1621 agcggcggca atcccgaccc tactttgtag gggagggcgc acttaccggt ttctcttcga
     1681 gaaactggcc taacggccac ccttcgggcg gtgcgctctc cgagggccat tgcatggagc
     1741 cgaaaagcaa aagcaacagc gaggcagcat ggcgatttat caccttacgg cgaaaaccgg
     1801 cagcaggtcg ggcggccaat cggccagggc caaggccgac tacatccagc gcgaaggcaa
     1861 gtatgcccgc gacatggatg aagtcttgca cgccgaatcc gggcacatgc cggagttcgt
     1921 cgagcggccc gccgactact gggatgctgc cgacctgtat gaacgcgcca atgggcggct
     1981 gttcaaggag gtcgaatttg ccctgccggt cgagctgacc ctcgaccagc agaaggcgct
     2041 ggcgtccgag ttcgcccagc acctgaccgg tgccgagcgc ctgccgtata cgctggccat
     2101 ccatgccggt ggcggcgaga acccgcactg ccacctgatg atctccgagc ggatcaatga
     2161 cggcatcgag cggcccgccg ctcagtggtt caagcggtac aacggcaaga ccccggagaa
     2221 gggcggggca cagaagaccg aagcgctcaa gcccaaggca tggcttgagc agacccgcga
     2281 ggcatgggcc gaccatgcca accgggcatt agagcgggct ggccacgacg cccgcattga
     2341 ccacagaaca cttgaggcgc agggcatcga gcgcctgccc ggtgttcacc tggggccgaa
     2401 cgtggtggag atggaaggcc ggggcatccg caccgaccgg gcagacgtgg ccctgaacat
     2461 cgacaccgcc aacgcccaga tcatcgactt acaggaatac cgggaggcaa tagaccatga
     2521 acgcaatcga cagagtgaag aaatccagag gcatcaacga gttagcggag cagatcgaac
     2581 cgctggccca gagcatggcg acactggccg acgaagcccg gcaggtcatg agccagaccc
     2641 agcaggccag cgaggcgcag gcggcggagt ggctgaaagc ccagcgccag acaggggcgg
     2701 catgggtgga gctggccaaa gagttgcggg aggtagccgc cgaggtgagc agcgccgcgc
     2761 agagcgcccg gagcgcgtcg cgggggtggc actggaagct atggctaacc gtgatgctgg
     2821 cttccatgat gcctacggtg gtgctgctga tcgcatcgtt gctcttgctc gacctgacgc
     2881 cactgacaac cgaggacggc tcgatctggc tgcgcttggt ggcccgatga agaacgacag
     2941 gactttgcag gccataggcc gacagctcaa ggccatgggc tgtgagcgct tcgatatcgg
     3001 cgtcagggac gccaccaccg gccagatgat gaaccgggaa tggtcagccg ccgaagtgct
     3061 ccagaacacg ccatggctca agcggatgaa tgcccagggc aatgacgtgt atatcaggcc
     3121 cgccgagcag gagcggcatg gtctggtgct ggtggacgac ctcagcgagt ttgacctgga
     3181 tgacatgaaa gccgagggcc gggagcctgc cctggtagtg gaaaccagcc cgaagaacta
     3241 tcaggcatgg gtcaaggtgg ccgacgccgc aggcggtgaa cttcgggggc agattgcccg
     3301 gacgctggcc agcgagtacg acgccgaccc ggccagcgcc gacagccgcc actatggccg
     3361 cttggcgggc ttcaccaacc gcaaggacaa gcacaccacc cgcgccggtt atcagccgtg
     3421 ggtgctgctg cgtgaatcca agggcaagac cgccaccgct ggcccggcgc tggtgcagca
     3481 ggctggccag cagatcgagc aggcccagcg gcagcaggag aaggcccgca ggctggccag
     3541 cctcgaactg cccgagcggc agcttagccg ccaccggcgc acggcgctgg acgagtaccg
     3601 cagcgagatg gccgggctgg tcaagcgctt cggtgatgac ctcagcaagt gcgactttat
     3661 cgccgcgcag aagctggcca gccggggccg cagtgccgag gaaatcggca aggccatggc
     3721 cgaggccagc ccagcgctgg cagagcgcaa gcccggccac gaagcggatt acatcgagcg
     3781 caccgtcagc aaggtcatgg gtctgcccag cgtccagctt gcgcgggccg agctggcacg
     3841 ggcaccggca ccccgccagc gaggcatgga caggggcggg ccagatttca gcatgtagtg
     3901 cttgcgttgg tactcacgcc tgttatacta tgagtactca cgcacagaag ggggttttat
     3961 ggaatacgaa aaaagcgctt cagggtcggt ctacctgatc aaaagtgaca agggctattg
     4021 gttgcccggt ggctttggtt atacgtcaaa caaggccgag gctggccgct tttcagtcgc
     4081 tgatatggcc agccttaacc ttgacggctg caccttgtcc ttgttccgcg aagacaagcc
     4141 tttcggcccc ggcaagtttc tcggtgactg atatgaaaga ccaaaaggac aagcagaccg
     4201 gcgacctgct ggccagccct gacgctgtac gccaagcgcg atatgccgag cgcatgaagg
     4261 ccaaagggat gcgtcagcgc aagttctggc tgaccgacga cgaatacgag gcgctgcgcg
     4321 agtgcctgga agaactcaga gcggcgcagg gcgggggtag tgaccccgcc agcgcctaac
     4381 caccaactgc ctgcaaagga ggcaatcaat ggctacccat aagcctatca atattctgga
     4441 ggcgttcgca gcagcgccgc caccgctgga ctacgttttg cccaacatgg tggccggtac
     4501 ggtcggggcg ctggtgtcgc ccggtggtgc cggtaaatcc atgctggccc tgcaactggc
     4561 cgcacagatt gcaggcgggc cggatctgct ggaggtgggc gaactgccca ccggcccggt
     4621 gatctacctg cccgccgaag acccgcccac cgccattcat caccgcctgc acgcccttgg
     4681 ggcgcacctc agcgccgagg aacggcaagc cgtggctgac ggcctgctga tccagccgct
     4741 gatcggcagc ctgcccaaca tcatggcccc ggagtggttc gacggcctca agcgcgccgc
     4801 cgagggccgc cgcctgatgg tgctggacac gctgcgccgg ttccacatcg aggaagaaaa
     4861 cgccagcggc cccatggccc aggtcatcgg tcgcatggag gccatcgccg ccgataccgg
     4921 gtgctctatc gtgttcctgc accatgccag caagggcgcg gccatgatgg gcgcaggcga
     4981 ccagcagcag gccagccggg gcagctcggt actggtcgat aacatccgct ggcagtccta
     5041 cctgtcgagc atgaccagcg ccgaggccga ggaatggggt gtggacgacg accagcgccg
     5101 gttcttcgtc cgcttcggtg tgagcaaggc caactatggc gcaccgttcg ctgatcggtg
     5161 gttcaggcgg catgacggcg gggtgctcaa gcccgccgtg ctggagaggc agcgcaagag
     5221 caagggggtg ccccgtggtg aagcctaaga acaagcacag cctcagccac gtccggcacg
     5281 acccggcgca ctgtctggcc cccggcctgt tccgtgccct caagcggggc gagcgcaagc
     5341 gcagcaagct ggacgtgacg tatgactacg gcgacggcaa gcggatcgag ttcagcggcc
     5401 cggagccgct gggcgctgat gatctgcgca tcctgcaagg gctggtggcc atggctgggc
     5461 ctaatggcct agtgcttggc ccggaaccca agaccgaagg cggacggcag ctccggctgt
     5521 tcctggaacc caagtgggag gccgtcaccg ctgatgccat ggtggtcaaa ggtagctatc
     5581 gggcgctggc aaaggaaatc ggggcagagg tcgatagtgg tggggcgctc aagcacatac
     5641 aggactgcat cgagcgcctt tggaaggtat ccatcatcgc ccagaatggc cgcaagcggc
     5701 aggggtttcg gctgctgtcg gagtacgcca gcgacgaggc ggacgggcgc ctgtacgtgg
     5761 ccctgaaccc cttgatcgcg caggccgtca tgggtggcgg ccagcatgtg cgcatcagca
     5821 tggacgaggt gcgggcgctg gacagcgaaa ccgcccgcct gctgcaccag cggctgtgtg
     5881 gctggatcga ccccggcaaa accggcaagg cttccataga taccttgtgc ggctatgtct
     5941 ggccgtcaga ggccagtggt tcgaccatgc gcaagcgccg ccagcgggtg cgcgaggcgt
     6001 tgccggagct ggtcgcgctg ggctggacgg taaccgagtt cgcggcgggc aagtacgaca
     6061 tcacccggcc caaggcggca ggctgactca cgttaaggga ttttggtcat gactgatcct
     6121 tcaactcagc aaaagttcga tttattcaac aaagccacgt tgtgtctcaa aatctctgat
     6181 gttacattgc acaagataaa aatatatcat catgaacaat aaaactgtct gcttacataa
     6241 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc tccaggccgc
     6301 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     6361 ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc
     6421 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     6481 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     6541 catggttact caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc
     6601 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     6661 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgcctcgct caggcgcaat
     6721 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     6781 ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg gattcagtcg
     6841 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     6901 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     6961 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     7021 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag
     7081 aattggttaa ttggttgtaa cacgcggccg ctcggtataa acgcagaaag gcccacccga
     7141 aggtgagcca gtgtgactct agtagagagc gttcaccgac aaacaacaga taaaacgaaa
     7201 ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggtgtact gcagtgcact
     7261 agtggttcgt aacatctctg taactgcttg agacgtggac tgttgtgata tgccatatat
     7321 gcagtggtgt tcgccggcgc tggttccggc tgtcttgctt agttgtgagt cgccaggcag
     7381 tcatcggtat gatctgtaca tgattcgtca gtgtgagcac cactgacgag cagatttcca
     7441 gctgacaaat gctctttccc taaactcccc ccataaaaaa acccgccgaa gcgggttttt
     7501 acgttatttg cggattaacg attacggatg gctcac
//
