LOCUS       sequence-208076-        7999 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BTK assembled broad-host-range plasmid encoding E2-Crimson protein 
            driven by PA3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7999)
  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 7999)
  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: pBTK570
FEATURES             Location/Qualifiers
     source          1..7999
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     oriT            2081..2168
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     rep_origin      2510..2904
                     /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             3637..4311
                     /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     4035..4054
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(4511..5302)
                     /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(5682..6533)
                     /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(6520..7359)
                     /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(7870..7999,1..842))
                     /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 tccgcttcgt ggccgggctt gcgctctgcc agcgctgggc tggcctcggc catggccttg
       61 ccgatttcct cggcactgcg gccccggctg gccagcttct gcgcggcgat aaagtcgcac
      121 ttgctgaggt catgaccgaa gcgcttgacc agcccggcca tctcgctgcg gtactcgtcc
      181 agcgccgtgc gccggtggcg gctaagctgc cgctcgggca gttcgaggct ggccagcctg
      241 cgggccttct cctgctgccg ctgggcctgc tcgatctgct ggccagcctg ctgcaccagc
      301 gccgggccag cggtggcggt cttgcccttg gattcacgca gcagcaccca cggctgataa
      361 ccggcgcggg tggtgtgctt gtccttgcgg ttggtgaagc ccgccaagcg gccatagtgg
      421 cggctgtcgg cgctggccgg gtcggcgtcg tactcgctgg ccagcgtccg ggcaatctgc
      481 ccccgaagtt caccgcctgc ggcgtcggcc accttgaccc atgcctgata gttcttcggg
      541 ctggtttcca ctaccagggc aggctcccgg ccctcggctt tcatgtcatc caggtcaaac
      601 tcgctgaggt cgtccaccag caccagacca tgccgctcct gctcggcggg cctgatatac
      661 acgtcattgc cctgggcatt catccgcttg agccatggcg tgttctggag cacttcggcg
      721 gctgaccatt cccggttcat catctggccg gtgggtgcgt ccctgacgcc gatatcgaag
      781 cgctcacagc ccatggcctt gagctgtcgg cctatggcct gcaaagtcct gtcgttcttc
      841 atcgggccac caagcgcagc cagatcgagc cgtcctcggt tgtcagtggc gtcaggtcga
      901 gcaagagcaa cgatgcgatc agcagcacca ccgtaggcat catggaagcc agcatcacgg
      961 ttagccatag cttccagtgc cacccccgcg acgcgctccg ggcgctctgc gcggcgctgc
     1021 tcacctcggc ggctacctcc cgcaactctt tggccagctc cacccatgcc gcccctgtct
     1081 ggcgctgggc tttcagccac tccgccgcct gcgcctcgct ggcctgcttg gtctggctca
     1141 tgacctgccg ggcttcgtcg gccagtgtcg ccatgctctg ggccagcggt tcgatctgct
     1201 ccgctaactc gttgatgcct ctggatttct tcactctgtc gattgcgttc atggtctatt
     1261 gcctcccggt attcctgtaa gtcgatgatc tgggcgttgg cggtgtcgat gttcagggcc
     1321 acgtctgccc ggtcggtgcg gatgccccgg ccttccatct ccaccacgtt cggccccagg
     1381 tgaacaccgg gcaggcgctc gatgccctgc gcctcaagtg ttctgtggtc aatgcgggcg
     1441 tcgtggccag cccgctctaa tgcccggttg gcatggtcgg cccatgcctc gcgggtctgc
     1501 tcaagccatg ccttgggctt gagcgcttcg gtcttctgtg ccccgccctt ctccggggtc
     1561 ttgccgttgt accgcttgaa ccactgagcg gcgggccgct cgatgccgtc attgatccgc
     1621 tcggagatca tcaggtggca gtgcgggttc tcgccgccac cggcatggat ggccagcgta
     1681 tacggcaggc gctcggcacc ggtcaggtgc tgggcgaact cggacgccag cgccttctgc
     1741 tggtcgaggg tcagctcgac cggcagggca aattcgacct ccttgaacag ccgcccattg
     1801 gcgcgttcat acaggtcggc agcatcccag tagtcggcgg gccgctcgac gaactccggc
     1861 atgtgcccgg attcggcgtg caagacttca tccatgtcgc gggcatactt gccttcgcgc
     1921 tggatgtagt cggccttggc cctggccgat tggccgcccg acctgctgcc ggttttcgcc
     1981 gtaaggtgat aaatcgccat gctgcctcgc tgttgctttt gcttttcggc tccatgcaat
     2041 ggccctcgga gagcgcaccg cccgaagggt ggccgttagg ccagtttctc gaagagaaac
     2101 cggtaagtgc gccctcccct acaaagtagg gtcgggattg ccgccgctgt gcctccatga
     2161 tagcctacga gacagcacat taacaatggg gtgtcaagat ggttaagggg agcaacaagg
     2221 cggcggatcg gctggccaag ctcgaagaac aacgagcgcg aatcaatgcc gaaattcagc
     2281 gggagcgggc aagggaacag cagcaagagc gcaagaacga aacaaggcgc aaggtgctgg
     2341 tgggggccat gattttggcc aaggtgaaca gcagcgagtg gccggaggat cggctcatgg
     2401 cggcaatgga tgcgtacctt gaacgcgacc acgaccgcgc cttgttcggt ctgccgccac
     2461 gccagaagga tgagccgggc tgaatgatcg accgagacag gccctgcggg gctgcacacg
     2521 cgcccccacc cttcgggtag ggggaaaggc cgctaaagcg gctaaaagcg ctccagcgta
     2581 tttctgcggg gtttggtgtg gggtttagcg ggctttgccc gcctttcccc ctgccgcgca
     2641 gcggtggggc ggtgtgtagc ctagcgcagc gaatagacca gctatccggc ctctggccgg
     2701 gcatattggg caagggcagc agcgccccac aagggcgctg ataaccgcgc ctagtggatt
     2761 attcttagat aatcatggat ggatttttcc aacaccccgc cagcccccgc ccctgctggg
     2821 tttgcaggtt tgggggcgtg acagttattg caggggttcg tgacagttat tgcagggggg
     2881 cgtgacagtt attgcagggg ttcgtgacag ttagtacggg agtgacgggc actggctggc
     2941 aatgtctagc aacggcaggc atttcggctg agggtaaaag aactttccgc taagcgatag
     3001 actgtatgta aacacagtat tgcaaggacg cggaacatgc ctcatgtggc ggccaggacg
     3061 gccagccggg atcgggatac tggtcgttac cagagccacc gacccgagca aacccttctc
     3121 tatcagatcg ttgacgagta ttacccggca ttcgctgcgc ttatggcaga gcagggaaag
     3181 gaattgccgg gctatgtgca acgggaattt gaagaatttc tccaatgcgg gcggctggag
     3241 catggctttc tacgggttcg ctgcgagtct tgccacgccg agcacctggt cgctttcaga
     3301 aatcaatcta aagtatatat gagtaaactt ggtctgacag gcccctgaat tcgcatctag
     3361 atggtagagc cacaaacagc cggtacaagc aacgatctcc aggaccatct gaatcatgcg
     3421 cggatgacac gaactcacga cggcgatcac agacattaac ccacagtaca gacactgcga
     3481 caacgtggca attcgtcgca ataccgtctc actgaactgg ccgataattg cagacgaacg
     3541 ggtgaaacaa aacggttgac aacatgaagt aaacacggta cgatgtacca catgaaacga
     3601 cagtgagtca atacagaaac agaggagata ttacatatgg atagcactga gaacgtcatc
     3661 aagcccttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag
     3721 atcgagggcg tgggcgaggg caagccctac gagggcaccc agaccgccaa gctgcaagtg
     3781 accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt cttctacggc
     3841 tccaaggcgt acatcaagca ccccgccgac atccccgact acctcaagca gtccttcccc
     3901 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc
     3961 caggactcct ccctgcagga cggcaccctc atctaccacg tgaagttcat cggcgtgaac
     4021 ttcccctccg acggccccgt aatgcagaag aagactctgg gctgggagcc ctccactgag
     4081 cgcaactacc cccgcgacgg cgtgctgaag ggcgagaacc acatggcgct gaagctgaag
     4141 ggcggcggcc actacctgtg tgagttcaag tccatctaca tggccaagaa gcccgtgaag
     4201 ctgcccggct accactacgt ggactacaag ctcgacatca cctcccacaa cgaggactac
     4261 accgtggtgg agcagtacga gcgcgccgag gcccgccacc acctgttcca gactcacggt
     4321 atggacgaat tgtacaagca cgacgaattg taaatccgta atcgttaatc cgcaaataac
     4381 gtaaaaaccc gcttcggcgg gtttttttat ggggggagtt tagggaaaga gcatttgtca
     4441 gctggatcag cagatgggaa ccggatactc ctcagcctcc agagtagcca atgagacgcc
     4501 gagcggccgc ttatttgccg actaccttgg tgatctcgcc tttcacgtag tggacaaatt
     4561 cttccaactg atctgcgcgc gaggccaagc gatcttcttc ttgtccaaga taagcctgtc
     4621 tagcttcaag tatgacgggc tgatactggg ccggcaggcg ctccattgcc cagtcggcag
     4681 cgacatcctt cggcgcgatt ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa
     4741 gcactacatt tcgctcatcg ccagcccagt cgggcggcga gttccatagc gttaaggttt
     4801 catttagcgc ctcaaataga tcctgttcag gaaccggatc aaagagttcc tccgccgctg
     4861 gacctaccaa ggcaacgcta tgttctcttg cttttgtcag caagatagcc agatcaatgt
     4921 cgatcgtggc tggctcgaag atacccgcaa gaatgtcatt gcgctgccat tctccaaatt
     4981 gcagttcgcg cttagctgga taacgccacg gaatgatgtc gtcgtgcaca acaatggtga
     5041 cttctacagc gcggagaatc tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt
     5101 tgatcaaagc tcgccgcgtt gtttcatcaa gccttacggt caccgtaacc agcaaatcaa
     5161 tatcactgtg tggcttcagg ccgccatcca ctgcggagcc gtacaaatgt acggccagca
     5221 acgtcggttc gagatggcgc tcgatgacgc caactacctc tgatagttga gtcgatactt
     5281 cggcgatcac cgcttccctc ataatgttta actttgtttt agggcgactg ccctgctgcg
     5341 taacatcgtt gctgctccat aacatcaaac atcgacccac ggcgtaacgc gcttgctgct
     5401 tggatgcccg aggcatagac tgtaccccaa aaaaacagtc ataacaagcc atgaaaaccg
     5461 ccactgcgcc gttaccaccg ctgcgttcgg tcaaggttct ggaccagttg cgtgagcgca
     5521 tacgctactt gcattacagc ttacgaaccg aacaggctta tgtccactgg gttcgtgcct
     5581 tcatccgttt ccacggtgtg cgtcacccgg caaccttggg tagcagcgaa gtcgaggcat
     5641 ttctgtcctg gctggtcatg accaaaatcc cttaacgtga gtcagcctgc cgccttgggc
     5701 cgggtgatgt cgtacttgcc cgccgcgaac tcggttaccg tccagcccag cgcgaccagc
     5761 tccggcaacg cctcgcgcac ccgcttgcgg cgcttgcgca tggtcgaacc actggcctct
     5821 gacggccaga catagccgca caaggtatct atggaagcct tgccggtttt gccggggtcg
     5881 atccagccac acagccgctg gtgcagcagg cgggcggttt cgctgtccag cgcccgcacc
     5941 tcgtccatgc tgatgcgcac atgctggccg ccacccatga cggcctgcgc gatcaagggg
     6001 ttcagggcca cgtacaggcg cccgtccgcc tcgtcgctgg cgtactccga cagcagccga
     6061 aacccctgcc gcttgcggcc attctgggcg atgatggata ccttccaaag gcgctcgatg
     6121 cagtcctgta tgtgcttgag cgccccacca ctatcgacct ctgccccgat ttcctttgcc
     6181 agcgcccgat agctaccttt gaccacatgg cattcagcgg tgacggcctc ccacttgggt
     6241 tccaggaaca gccggagctg ccgtccgcct tcggtcttgg gttccgggcc aagcactagg
     6301 ccattaggcc cagccatggc caccagccct tgcaggatgc gcagatcatc agcgcccagc
     6361 ggctccgggc cgctgaactc gatccgcttg ccgtcgccgt agtcatacgt cacgtccagc
     6421 ttgctgcgct tgcgctcgcc ccgcttgagg gcacggaaca ggccgggggc cagacagtgc
     6481 gccgggtcgt gccggacgtg gctgaggctg tgcttgttct taggcttcac cacggggcac
     6541 ccccttgctc ttgcgctgcc tctccagcac ggcgggcttg agcaccccgc cgtcatgccg
     6601 cctgaaccac cgatcagcga acggtgcgcc atagttggcc ttgctcacac cgaagcggac
     6661 gaagaaccgg cgctggtcgt cgtccacacc ccattcctcg gcctcggcgc tggtcatgct
     6721 cgacaggtag gactgccagc ggatgttatc gaccagtacc gagctgcccc ggctggcctg
     6781 ctgctggtcg cctgcgccca tcatggccgc gcccttgctg gcatggtgca ggaacacgat
     6841 agagcacccg gtatcggcgg cgatggcctc catgcgaccg atgacctggg ccatggggcc
     6901 gctggcgttt tcttcctcga tgtggaaccg gcgcagcgtg tccagcacca tcaggcggcg
     6961 gccctcggcg gcgcgcttga ggccgtcgaa ccactccggg gccatgatgt tgggcaggct
     7021 gccgatcagc ggctggatca gcaggccgtc agccacggct tgccgttcct cggcgctgag
     7081 gtgcgcccca agggcgtgca ggcggtgatg aatggcggtg ggcgggtctt cggcgggcag
     7141 gtagatcacc gggccggtgg gcagttcgcc cacctccagc agatccggcc cgcctgcaat
     7201 ctgtgcggcc agttgcaggg ccagcatgga tttaccggca ccaccgggcg acaccagcgc
     7261 cccgaccgta ccggccacca tgttgggcaa aacgtagtcc agcggtggcg gcgctgctgc
     7321 gaacgcctcc agaatattga taggcttatg ggtagccatt gattgcctcc tttgcaggca
     7381 gttggtggtt aggcgctggc ggggtcacta cccccgccct gcgccgctct gagttcttcc
     7441 aggcactcgc gcagcgcctc gtattcgtcg tcggtcagcc agaacttgcg ctgacgcatc
     7501 cctttggcct tcatgcgctc ggcatatcgc gcttggcgta cagcgtcagg gctggccagc
     7561 aggtcgccgg tctgcttgtc cttttggtct ttcatatcag tcaccgagaa acttgccggg
     7621 gccgaaaggc ttgtcttcgc ggaacaagga caaggtgcag ccgtcaaggt taaggctggc
     7681 catatcagcg actgaaaagc ggccagcctc ggccttgttt gacgtataac caaagccacc
     7741 gggcaaccaa tagcccttgt cacttttgat caggtagacc gaccctgaag cgcttttttc
     7801 gtattccata aaaccccctt ctgtgcgtga gtactcatag tataacaggc gtgagtacca
     7861 acgcaagcac tacatgctga aatctggccc gcccctgtcc atgcctcgct ggcggggtgc
     7921 cggtgcccgt gccagctcgg cccgcgcaag ctggacgctg ggcagaccca tgaccttgct
     7981 gacggtgcgc tcgatgtaa
//
