LOCUS       sequence-218139-        7988 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BTK assembled broad-host-range plasmid encoding E2-Crimson protein 
            driven by CP25 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7988)
  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 7988)
  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: pBTK569
FEATURES             Location/Qualifiers
     source          1..7988
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             617..1468
                     /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             1848..2639
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     CDS             complement(2839..3513)
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGMGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHM
                     ALKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(3096..3115)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     rep_origin      4235..4629
                     /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            4971..5058
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     CDS             6297..7268
                     /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             join(7779..7988,1..630)
                     /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"
ORIGIN
        1 gtgatctacc tgcccgccga agacccgccc accgccattc atcaccgcct gcacgccctt
       61 ggggcgcacc tcagcgccga ggaacggcaa gccgtggctg acggcctgct gatccagccg
      121 ctgatcggca gcctgcccaa catcatggcc ccggagtggt tcgacggcct caagcgcgcc
      181 gccgagggcc gccgcctgat ggtgctggac acgctgcgcc ggttccacat cgaggaagaa
      241 aacgccagcg gccccatggc ccaggtcatc ggtcgcatgg aggccatcgc cgccgatacc
      301 gggtgctcta tcgtgttcct gcaccatgcc agcaagggcg cggccatgat gggcgcaggc
      361 gaccagcagc aggccagccg gggcagctcg gtactggtcg ataacatccg ctggcagtcc
      421 tacctgtcga gcatgaccag cgccgaggcc gaggaatggg gtgtggacga cgaccagcgc
      481 cggttcttcg tccgcttcgg tgtgagcaag gccaactatg gcgcaccgtt cgctgatcgg
      541 tggttcaggc ggcatgacgg cggggtgctc aagcccgccg tgctggagag gcagcgcaag
      601 agcaaggggg tgccccgtgg tgaagcctaa gaacaagcac agcctcagcc acgtccggca
      661 cgacccggcg cactgtctgg cccccggcct gttccgtgcc ctcaagcggg gcgagcgcaa
      721 gcgcagcaag ctggacgtga cgtatgacta cggcgacggc aagcggatcg agttcagcgg
      781 cccggagccg ctgggcgctg atgatctgcg catcctgcaa gggctggtgg ccatggctgg
      841 gcctaatggc ctagtgcttg gcccggaacc caagaccgaa ggcggacggc agctccggct
      901 gttcctggaa cccaagtggg aggccgtcac cgctgatgcc atggtggtca aaggtagcta
      961 tcgggcgctg gcaaaggaaa tcggggcaga ggtcgatagt ggtggggcgc tcaagcacat
     1021 acaggactgc atcgagcgcc tttggaaggt atccatcatc gcccagaatg gccgcaagcg
     1081 gcaggggttt cggctgctgt cggagtacgc cagcgacgag gcggacgggc gcctgtacgt
     1141 ggccctgaac cccttgatcg cgcaggccgt catgggtggc ggccagcatg tgcgcatcag
     1201 catggacgag gtgcgggcgc tggacagcga aaccgcccgc ctgctgcacc agcggctgtg
     1261 tggctggatc gaccccggca aaaccggcaa ggcttccata gataccttgt gcggctatgt
     1321 ctggccgtca gaggccagtg gttcgaccat gcgcaagcgc cgccagcggg tgcgcgaggc
     1381 gttgccggag ctggtcgcgc tgggctggac ggtaaccgag ttcgcggcgg gcaagtacga
     1441 catcacccgg cccaaggcgg caggctgact cacgttaagg gattttggtc atgaccagcc
     1501 aggacagaaa tgcctcgact tcgctgctac ccaaggttgc cgggtgacgc acaccgtgga
     1561 aacggatgaa ggcacgaacc cagtggacat aagcctgttc ggttcgtaag ctgtaatgca
     1621 agtagcgtat gcgctcacgc aactggtcca gaaccttgac cgaacgcagc ggtggtaacg
     1681 gcgcagtggc ggttttcatg gcttgttatg actgtttttt tggggtacag tctatgcctc
     1741 gggcatccaa gcagcaagcg cgttacgccg tgggtcgatg tttgatgtta tggagcagca
     1801 acgatgttac gcagcagggc agtcgcccta aaacaaagtt aaacattatg agggaagcgg
     1861 tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag cgccatctcg
     1921 aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc ctgaagccac
     1981 acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca acgcggcgag
     2041 ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag attctccgcg
     2101 ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat ccagctaagc
     2161 gcgaactgca atttggagaa tggcagcgca atgacattct tgcgggtatc ttcgagccag
     2221 ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat agcgttgcct
     2281 tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat ctatttgagg
     2341 cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc gatgagcgaa
     2401 atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa atcgcgccga
     2461 aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag cccgtcatac
     2521 ttgaagctag acaggcttat cttggacaag aagaagatcg cttggcctcg cgcgcagatc
     2581 agttggaaga atttgtccac tacgtgaaag gcgagatcac caaggtagtc ggcaaataag
     2641 cggccgctcg gcgtctcatt ggctactctg gaggctgagg agtatccggt tcccatctgc
     2701 tgatccagct gacaaatgct ctttccctaa actcccccca taaaaaaacc cgccgaagcg
     2761 ggtttttacg ttatttgcgg attaacgatt acggatttac aattcgtcgt gcttgtacaa
     2821 ttcgtccata ccgtgagtct ggaacaggtg gtggcgggcc tcggcgcgct cgtactgctc
     2881 caccacggtg tagtcctcgt tgtgggaggt gatgtcgagc ttgtagtcca cgtagtggta
     2941 gccgggcagc ttcacgggct tcttggccat gtagatggac ttgaactcac acaggtagtg
     3001 gccgccgccc ttcagcttca gcgccatgtg gttctcgccc ttcagcacgc cgtcgcgggg
     3061 gtagttgcgc tcagtggagg gctcccagcc cagagtcttc ttctgcatta cggggccgtc
     3121 ggaggggaag ttcacgccga tgaacttcac gtggtagatg agggtgccgt cctgcaggga
     3181 ggagtcctgg gtcacggtca ccacgccgcc gtcctcgaag ttcatcacgc gctcccactt
     3241 gaagccctcg gggaaggact gcttgaggta gtcggggatg tcggcggggt gcttgatgta
     3301 cgccttggag ccgtagaaga actgggggga caggatgtcc caggcgaagg gcagggggcc
     3361 gcccttggtc acttgcagct tggcggtctg ggtgccctcg tagggcttgc cctcgcccat
     3421 gccctcgatc tcgaactcgt ggccgttcac ggagccctcc atgtgcacct tgaagcgcat
     3481 gaagggcttg atgacgttct cagtgctatc catatgtaat atctcctctg tttctgtata
     3541 acagtactat gtgattatac cagccccctc actacatgtc aagaataaac tgccaaagcg
     3601 ttcgtctgca attatcggcc agttcagtga gacggtattg cgacgaattg ccacgttgtc
     3661 gcagtgtctg tactgtgggt taatgtctgt gatcgccgtc gtgagttcgt gtcatccgcg
     3721 catgattcag atggtcctgg agatcgttgc ttgtaccggc tgtttgtggc tctaccatct
     3781 agatgcgaat tcaggggcct gtcagaccaa gtttactcat atatacttta gattgatttc
     3841 tgaaagcgac caggtgctcg gcgtggcaag actcgcagcg aacccgtaga aagccatgct
     3901 ccagccgccc gcattggaga aattcttcaa attcccgttg cacatagccc ggcaattcct
     3961 ttccctgctc tgccataagc gcagcgaatg ccgggtaata ctcgtcaacg atctgataga
     4021 gaagggtttg ctcgggtcgg tggctctggt aacgaccagt atcccgatcc cggctggccg
     4081 tcctggccgc cacatgaggc atgttccgcg tccttgcaat actgtgttta catacagtct
     4141 atcgcttagc ggaaagttct tttaccctca gccgaaatgc ctgccgttgc tagacattgc
     4201 cagccagtgc ccgtcactcc cgtactaact gtcacgaacc cctgcaataa ctgtcacgcc
     4261 cccctgcaat aactgtcacg aacccctgca ataactgtca cgcccccaaa cctgcaaacc
     4321 cagcaggggc gggggctggc ggggtgttgg aaaaatccat ccatgattat ctaagaataa
     4381 tccactaggc gcggttatca gcgcccttgt ggggcgctgc tgcccttgcc caatatgccc
     4441 ggccagaggc cggatagctg gtctattcgc tgcgctaggc tacacaccgc cccaccgctg
     4501 cgcggcaggg ggaaaggcgg gcaaagcccg ctaaacccca caccaaaccc cgcagaaata
     4561 cgctggagcg cttttagccg ctttagcggc ctttccccct acccgaaggg tgggggcgcg
     4621 tgtgcagccc cgcagggcct gtctcggtcg atcattcagc ccggctcatc cttctggcgt
     4681 ggcggcagac cgaacaaggc gcggtcgtgg tcgcgttcaa ggtacgcatc cattgccgcc
     4741 atgagccgat cctccggcca ctcgctgctg ttcaccttgg ccaaaatcat ggcccccacc
     4801 agcaccttgc gccttgtttc gttcttgcgc tcttgctgct gttcccttgc ccgcacccgc
     4861 tgaatttcgg cattgattcg cgctcgttgt tcttcgagct tggccagccg atccgccgcc
     4921 ttgttgctcc ccttaaccat cttgacaccc cattgttaat gtgctgtctc gtaggctatc
     4981 atggaggcac agcggcggca atcccgaccc tactttgtag gggagggcgc acttaccggt
     5041 ttctcttcga gaaactggcc taacggccac ccttcgggcg gtgcgctctc cgagggccat
     5101 tgcatggagc cgaaaagcaa aagcaacagc gaggcagcat ggcgatttat caccttacgg
     5161 cgaaaaccgg cagcaggtcg ggcggccaat cggccagggc caaggccgac tacatccagc
     5221 gcgaaggcaa gtatgcccgc gacatggatg aagtcttgca cgccgaatcc gggcacatgc
     5281 cggagttcgt cgagcggccc gccgactact gggatgctgc cgacctgtat gaacgcgcca
     5341 atgggcggct gttcaaggag gtcgaatttg ccctgccggt cgagctgacc ctcgaccagc
     5401 agaaggcgct ggcgtccgag ttcgcccagc acctgaccgg tgccgagcgc ctgccgtata
     5461 cgctggccat ccatgccggt ggcggcgaga acccgcactg ccacctgatg atctccgagc
     5521 ggatcaatga cggcatcgag cggcccgccg ctcagtggtt caagcggtac aacggcaaga
     5581 ccccggagaa gggcggggca cagaagaccg aagcgctcaa gcccaaggca tggcttgagc
     5641 agacccgcga ggcatgggcc gaccatgcca accgggcatt agagcgggct ggccacgacg
     5701 cccgcattga ccacagaaca cttgaggcgc agggcatcga gcgcctgccc ggtgttcacc
     5761 tggggccgaa cgtggtggag atggaaggcc ggggcatccg caccgaccgg gcagacgtgg
     5821 ccctgaacat cgacaccgcc aacgcccaga tcatcgactt acaggaatac cgggaggcaa
     5881 tagaccatga acgcaatcga cagagtgaag aaatccagag gcatcaacga gttagcggag
     5941 cagatcgaac cgctggccca gagcatggcg acactggccg acgaagcccg gcaggtcatg
     6001 agccagaccc agcaggccag cgaggcgcag gcggcggagt ggctgaaagc ccagcgccag
     6061 acaggggcgg catgggtgga gctggccaaa gagttgcggg aggtagccgc cgaggtgagc
     6121 agcgccgcgc agagcgcccg gagcgcgtcg cgggggtggc actggaagct atggctaacc
     6181 gtgatgctgg cttccatgat gcctacggtg gtgctgctga tcgcatcgtt gctcttgctc
     6241 gacctgacgc cactgacaac cgaggacggc tcgatctggc tgcgcttggt ggcccgatga
     6301 agaacgacag gactttgcag gccataggcc gacagctcaa ggccatgggc tgtgagcgct
     6361 tcgatatcgg cgtcagggac gccaccaccg gccagatgat gaaccgggaa tggtcagccg
     6421 ccgaagtgct ccagaacacg ccatggctca agcggatgaa tgcccagggc aatgacgtgt
     6481 atatcaggcc cgccgagcag gagcggcatg gtctggtgct ggtggacgac ctcagcgagt
     6541 ttgacctgga tgacatgaaa gccgagggcc gggagcctgc cctggtagtg gaaaccagcc
     6601 cgaagaacta tcaggcatgg gtcaaggtgg ccgacgccgc aggcggtgaa cttcgggggc
     6661 agattgcccg gacgctggcc agcgagtacg acgccgaccc ggccagcgcc gacagccgcc
     6721 actatggccg cttggcgggc ttcaccaacc gcaaggacaa gcacaccacc cgcgccggtt
     6781 atcagccgtg ggtgctgctg cgtgaatcca agggcaagac cgccaccgct ggcccggcgc
     6841 tggtgcagca ggctggccag cagatcgagc aggcccagcg gcagcaggag aaggcccgca
     6901 ggctggccag cctcgaactg cccgagcggc agcttagccg ccaccggcgc acggcgctgg
     6961 acgagtaccg cagcgagatg gccgggctgg tcaagcgctt cggtgatgac ctcagcaagt
     7021 gcgactttat cgccgcgcag aagctggcca gccggggccg cagtgccgag gaaatcggca
     7081 aggccatggc cgaggccagc ccagcgctgg cagagcgcaa gcccggccac gaagcggatt
     7141 acatcgagcg caccgtcagc aaggtcatgg gtctgcccag cgtccagctt gcgcgggccg
     7201 agctggcacg ggcaccggca ccccgccagc gaggcatgga caggggcggg ccagatttca
     7261 gcatgtagtg cttgcgttgg tactcacgcc tgttatacta tgagtactca cgcacagaag
     7321 ggggttttat ggaatacgaa aaaagcgctt cagggtcggt ctacctgatc aaaagtgaca
     7381 agggctattg gttgcccggt ggctttggtt atacgtcaaa caaggccgag gctggccgct
     7441 tttcagtcgc tgatatggcc agccttaacc ttgacggctg caccttgtcc ttgttccgcg
     7501 aagacaagcc tttcggcccc ggcaagtttc tcggtgactg atatgaaaga ccaaaaggac
     7561 aagcagaccg gcgacctgct ggccagccct gacgctgtac gccaagcgcg atatgccgag
     7621 cgcatgaagg ccaaagggat gcgtcagcgc aagttctggc tgaccgacga cgaatacgag
     7681 gcgctgcgcg agtgcctgga agaactcaga gcggcgcagg gcgggggtag tgaccccgcc
     7741 agcgcctaac caccaactgc ctgcaaagga ggcaatcaat ggctacccat aagcctatca
     7801 atattctgga ggcgttcgca gcagcgccgc caccgctgga ctacgttttg cccaacatgg
     7861 tggccggtac ggtcggggcg ctggtgtcgc ccggtggtgc cggtaaatcc atgctggccc
     7921 tgcaactggc cgcacagatt gcaggcgggc cggatctgct ggaggtgggc gaactgccca
     7981 ccggcccg
//
