LOCUS       sequence-208075-        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"
     oriT            2090..2177
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     rep_origin      2519..2913
                     /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)"
     CDS             3635..4309
                     /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="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHM
                     ALKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     4033..4052
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(4509..5300)
                     /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(5680..6531)
                     /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(6518..7357)
                     /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(7868..7988,1..851))
                     /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 tcgatgtaat ccgcttcgtg gccgggcttg cgctctgcca gcgctgggct ggcctcggcc
       61 atggccttgc cgatttcctc ggcactgcgg ccccggctgg ccagcttctg cgcggcgata
      121 aagtcgcact tgctgaggtc atgaccgaag cgcttgacca gcccggccat ctcgctgcgg
      181 tactcgtcca gcgccgtgcg ccggtggcgg ctaagctgcc gctcgggcag ttcgaggctg
      241 gccagcctgc gggccttctc ctgctgccgc tgggcctgct cgatctgctg gccagcctgc
      301 tgcaccagcg ccgggccagc ggtggcggtc ttgcccttgg attcacgcag cagcacccac
      361 ggctgataac cggcgcgggt ggtgtgcttg tccttgcggt tggtgaagcc cgccaagcgg
      421 ccatagtggc ggctgtcggc gctggccggg tcggcgtcgt actcgctggc cagcgtccgg
      481 gcaatctgcc cccgaagttc accgcctgcg gcgtcggcca ccttgaccca tgcctgatag
      541 ttcttcgggc tggtttccac taccagggca ggctcccggc cctcggcttt catgtcatcc
      601 aggtcaaact cgctgaggtc gtccaccagc accagaccat gccgctcctg ctcggcgggc
      661 ctgatataca cgtcattgcc ctgggcattc atccgcttga gccatggcgt gttctggagc
      721 acttcggcgg ctgaccattc ccggttcatc atctggccgg tgggtgcgtc cctgacgccg
      781 atatcgaagc gctcacagcc catggccttg agctgtcggc ctatggcctg caaagtcctg
      841 tcgttcttca tcgggccacc aagcgcagcc agatcgagcc gtcctcggtt gtcagtggcg
      901 tcaggtcgag caagagcaac gatgcgatca gcagcaccac cgtaggcatc atggaagcca
      961 gcatcacggt tagccatagc ttccagtgcc acccccgcga cgcgctccgg gcgctctgcg
     1021 cggcgctgct cacctcggcg gctacctccc gcaactcttt ggccagctcc acccatgccg
     1081 cccctgtctg gcgctgggct ttcagccact ccgccgcctg cgcctcgctg gcctgcttgg
     1141 tctggctcat gacctgccgg gcttcgtcgg ccagtgtcgc catgctctgg gccagcggtt
     1201 cgatctgctc cgctaactcg ttgatgcctc tggatttctt cactctgtcg attgcgttca
     1261 tggtctattg cctcccggta ttcctgtaag tcgatgatct gggcgttggc ggtgtcgatg
     1321 ttcagggcca cgtctgcccg gtcggtgcgg atgccccggc cttccatctc caccacgttc
     1381 ggccccaggt gaacaccggg caggcgctcg atgccctgcg cctcaagtgt tctgtggtca
     1441 atgcgggcgt cgtggccagc ccgctctaat gcccggttgg catggtcggc ccatgcctcg
     1501 cgggtctgct caagccatgc cttgggcttg agcgcttcgg tcttctgtgc cccgcccttc
     1561 tccggggtct tgccgttgta ccgcttgaac cactgagcgg cgggccgctc gatgccgtca
     1621 ttgatccgct cggagatcat caggtggcag tgcgggttct cgccgccacc ggcatggatg
     1681 gccagcgtat acggcaggcg ctcggcaccg gtcaggtgct gggcgaactc ggacgccagc
     1741 gccttctgct ggtcgagggt cagctcgacc ggcagggcaa attcgacctc cttgaacagc
     1801 cgcccattgg cgcgttcata caggtcggca gcatcccagt agtcggcggg ccgctcgacg
     1861 aactccggca tgtgcccgga ttcggcgtgc aagacttcat ccatgtcgcg ggcatacttg
     1921 ccttcgcgct ggatgtagtc ggccttggcc ctggccgatt ggccgcccga cctgctgccg
     1981 gttttcgccg taaggtgata aatcgccatg ctgcctcgct gttgcttttg cttttcggct
     2041 ccatgcaatg gccctcggag agcgcaccgc ccgaagggtg gccgttaggc cagtttctcg
     2101 aagagaaacc ggtaagtgcg ccctccccta caaagtaggg tcgggattgc cgccgctgtg
     2161 cctccatgat agcctacgag acagcacatt aacaatgggg tgtcaagatg gttaagggga
     2221 gcaacaaggc ggcggatcgg ctggccaagc tcgaagaaca acgagcgcga atcaatgccg
     2281 aaattcagcg ggagcgggca agggaacagc agcaagagcg caagaacgaa acaaggcgca
     2341 aggtgctggt gggggccatg attttggcca aggtgaacag cagcgagtgg ccggaggatc
     2401 ggctcatggc ggcaatggat gcgtaccttg aacgcgacca cgaccgcgcc ttgttcggtc
     2461 tgccgccacg ccagaaggat gagccgggct gaatgatcga ccgagacagg ccctgcgggg
     2521 ctgcacacgc gcccccaccc ttcgggtagg gggaaaggcc gctaaagcgg ctaaaagcgc
     2581 tccagcgtat ttctgcgggg tttggtgtgg ggtttagcgg gctttgcccg cctttccccc
     2641 tgccgcgcag cggtggggcg gtgtgtagcc tagcgcagcg aatagaccag ctatccggcc
     2701 tctggccggg catattgggc aagggcagca gcgccccaca agggcgctga taaccgcgcc
     2761 tagtggatta ttcttagata atcatggatg gatttttcca acaccccgcc agcccccgcc
     2821 cctgctgggt ttgcaggttt gggggcgtga cagttattgc aggggttcgt gacagttatt
     2881 gcaggggggc gtgacagtta ttgcaggggt tcgtgacagt tagtacggga gtgacgggca
     2941 ctggctggca atgtctagca acggcaggca tttcggctga gggtaaaaga actttccgct
     3001 aagcgataga ctgtatgtaa acacagtatt gcaaggacgc ggaacatgcc tcatgtggcg
     3061 gccaggacgg ccagccggga tcgggatact ggtcgttacc agagccaccg acccgagcaa
     3121 acccttctct atcagatcgt tgacgagtat tacccggcat tcgctgcgct tatggcagag
     3181 cagggaaagg aattgccggg ctatgtgcaa cgggaatttg aagaatttct ccaatgcggg
     3241 cggctggagc atggctttct acgggttcgc tgcgagtctt gccacgccga gcacctggtc
     3301 gctttcagaa atcaatctaa agtatatatg agtaaacttg gtctgacagg cccctgaatt
     3361 cgcatctaga tggtagagcc acaaacagcc ggtacaagca acgatctcca ggaccatctg
     3421 aatcatgcgc ggatgacacg aactcacgac ggcgatcaca gacattaacc cacagtacag
     3481 acactgcgac aacgtggcaa ttcgtcgcaa taccgtctca ctgaactggc cgataattgc
     3541 agacgaacgc tttggcagtt tattcttgac atgtagtgag ggggctggta taatcacata
     3601 gtactgttat acagaaacag aggagatatt acatatggat agcactgaga acgtcatcaa
     3661 gcccttcatg cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat
     3721 cgagggcgtg ggcgagggca agccctacga gggcacccag accgccaagc tgcaagtgac
     3781 caagggcggc cccctgccct tcgcctggga catcctgtcc ccccagttct tctacggctc
     3841 caaggcgtac atcaagcacc ccgccgacat ccccgactac ctcaagcagt ccttccccga
     3901 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca
     3961 ggactcctcc ctgcaggacg gcaccctcat ctaccacgtg aagttcatcg gcgtgaactt
     4021 cccctccgac ggccccgtaa tgcagaagaa gactctgggc tgggagccct ccactgagcg
     4081 caactacccc cgcgacggcg tgctgaaggg cgagaaccac atggcgctga agctgaaggg
     4141 cggcggccac tacctgtgtg agttcaagtc catctacatg gccaagaagc ccgtgaagct
     4201 gcccggctac cactacgtgg actacaagct cgacatcacc tcccacaacg aggactacac
     4261 cgtggtggag cagtacgagc gcgccgaggc ccgccaccac ctgttccaga ctcacggtat
     4321 ggacgaattg tacaagcacg acgaattgta aatccgtaat cgttaatccg caaataacgt
     4381 aaaaacccgc ttcggcgggt ttttttatgg ggggagttta gggaaagagc atttgtcagc
     4441 tggatcagca gatgggaacc ggatactcct cagcctccag agtagccaat gagacgccga
     4501 gcggccgctt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct
     4561 tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta
     4621 gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg
     4681 acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc
     4741 actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca
     4801 tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga
     4861 cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg
     4921 atcgtggctg gctcgaagat acccgcaaga atgtcattgc gctgccattc tccaaattgc
     4981 agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact
     5041 tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg
     5101 atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata
     5161 tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac
     5221 gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg
     5281 gcgatcaccg cttccctcat aatgtttaac tttgttttag ggcgactgcc ctgctgcgta
     5341 acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg
     5401 gatgcccgag gcatagactg taccccaaaa aaacagtcat aacaagccat gaaaaccgcc
     5461 actgcgccgt taccaccgct gcgttcggtc aaggttctgg accagttgcg tgagcgcata
     5521 cgctacttgc attacagctt acgaaccgaa caggcttatg tccactgggt tcgtgccttc
     5581 atccgtttcc acggtgtgcg tcacccggca accttgggta gcagcgaagt cgaggcattt
     5641 ctgtcctggc tggtcatgac caaaatccct taacgtgagt cagcctgccg ccttgggccg
     5701 ggtgatgtcg tacttgcccg ccgcgaactc ggttaccgtc cagcccagcg cgaccagctc
     5761 cggcaacgcc tcgcgcaccc gcttgcggcg cttgcgcatg gtcgaaccac tggcctctga
     5821 cggccagaca tagccgcaca aggtatctat ggaagccttg ccggttttgc cggggtcgat
     5881 ccagccacac agccgctggt gcagcaggcg ggcggtttcg ctgtccagcg cccgcacctc
     5941 gtccatgctg atgcgcacat gctggccgcc acccatgacg gcctgcgcga tcaaggggtt
     6001 cagggccacg tacaggcgcc cgtccgcctc gtcgctggcg tactccgaca gcagccgaaa
     6061 cccctgccgc ttgcggccat tctgggcgat gatggatacc ttccaaaggc gctcgatgca
     6121 gtcctgtatg tgcttgagcg ccccaccact atcgacctct gccccgattt cctttgccag
     6181 cgcccgatag ctacctttga ccacatggca ttcagcggtg acggcctccc acttgggttc
     6241 caggaacagc cggagctgcc gtccgccttc ggtcttgggt tccgggccaa gcactaggcc
     6301 attaggccca gccatggcca ccagcccttg caggatgcgc agatcatcag cgcccagcgg
     6361 ctccgggccg ctgaactcga tccgcttgcc gtcgccgtag tcatacgtca cgtccagctt
     6421 gctgcgcttg cgctcgcccc gcttgagggc acggaacagg ccgggggcca gacagtgcgc
     6481 cgggtcgtgc cggacgtggc tgaggctgtg cttgttctta ggcttcacca cggggcaccc
     6541 ccttgctctt gcgctgcctc tccagcacgg cgggcttgag caccccgccg tcatgccgcc
     6601 tgaaccaccg atcagcgaac ggtgcgccat agttggcctt gctcacaccg aagcggacga
     6661 agaaccggcg ctggtcgtcg tccacacccc attcctcggc ctcggcgctg gtcatgctcg
     6721 acaggtagga ctgccagcgg atgttatcga ccagtaccga gctgccccgg ctggcctgct
     6781 gctggtcgcc tgcgcccatc atggccgcgc ccttgctggc atggtgcagg aacacgatag
     6841 agcacccggt atcggcggcg atggcctcca tgcgaccgat gacctgggcc atggggccgc
     6901 tggcgttttc ttcctcgatg tggaaccggc gcagcgtgtc cagcaccatc aggcggcggc
     6961 cctcggcggc gcgcttgagg ccgtcgaacc actccggggc catgatgttg ggcaggctgc
     7021 cgatcagcgg ctggatcagc aggccgtcag ccacggcttg ccgttcctcg gcgctgaggt
     7081 gcgccccaag ggcgtgcagg cggtgatgaa tggcggtggg cgggtcttcg gcgggcaggt
     7141 agatcaccgg gccggtgggc agttcgccca cctccagcag atccggcccg cctgcaatct
     7201 gtgcggccag ttgcagggcc agcatggatt taccggcacc accgggcgac accagcgccc
     7261 cgaccgtacc ggccaccatg ttgggcaaaa cgtagtccag cggtggcggc gctgctgcga
     7321 acgcctccag aatattgata ggcttatggg tagccattga ttgcctcctt tgcaggcagt
     7381 tggtggttag gcgctggcgg ggtcactacc cccgccctgc gccgctctga gttcttccag
     7441 gcactcgcgc agcgcctcgt attcgtcgtc ggtcagccag aacttgcgct gacgcatccc
     7501 tttggccttc atgcgctcgg catatcgcgc ttggcgtaca gcgtcagggc tggccagcag
     7561 gtcgccggtc tgcttgtcct tttggtcttt catatcagtc accgagaaac ttgccggggc
     7621 cgaaaggctt gtcttcgcgg aacaaggaca aggtgcagcc gtcaaggtta aggctggcca
     7681 tatcagcgac tgaaaagcgg ccagcctcgg ccttgtttga cgtataacca aagccaccgg
     7741 gcaaccaata gcccttgtca cttttgatca ggtagaccga ccctgaagcg cttttttcgt
     7801 attccataaa acccccttct gtgcgtgagt actcatagta taacaggcgt gagtaccaac
     7861 gcaagcacta catgctgaaa tctggcccgc ccctgtccat gcctcgctgg cggggtgccg
     7921 gtgcccgtgc cagctcggcc cgcgcaagct ggacgctggg cagacccatg accttgctga
     7981 cggtgcgc
//
