LOCUS       sequence-218140-        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"
     CDS             617..1291
                     /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     1015..1034
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(1491..2282)
                     /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(2662..3513)
                     /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             complement(3500..4339)
                     /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(4850..5821)
                     /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"
     oriT            7060..7147
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     rep_origin      7489..7883
                     /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)"
ORIGIN
        1 tgcaaggacg cggaacatgc ctcatgtggc ggccaggacg gccagccggg atcgggatac
       61 tggtcgttac cagagccacc gacccgagca aacccttctc tatcagatcg ttgacgagta
      121 ttacccggca ttcgctgcgc ttatggcaga gcagggaaag gaattgccgg gctatgtgca
      181 acgggaattt gaagaatttc tccaatgcgg gcggctggag catggctttc tacgggttcg
      241 ctgcgagtct tgccacgccg agcacctggt cgctttcaga aatcaatcta aagtatatat
      301 gagtaaactt ggtctgacag gcccctgaat tcgcatctag atggtagagc cacaaacagc
      361 cggtacaagc aacgatctcc aggaccatct gaatcatgcg cggatgacac gaactcacga
      421 cggcgatcac agacattaac ccacagtaca gacactgcga caacgtggca attcgtcgca
      481 ataccgtctc actgaactgg ccgataattg cagacgaacg ggtgaaacaa aacggttgac
      541 aacatgaagt aaacacggta cgatgtacca catgaaacga cagtgagtca atacagaaac
      601 agaggagata ttacatatgg atagcactga gaacgtcatc aagcccttca tgcgcttcaa
      661 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg tgggcgaggg
      721 caagccctac gagggcaccc agaccgccaa gctgcaagtg accaagggcg gccccctgcc
      781 cttcgcctgg gacatcctgt ccccccagtt cttctacggc tccaaggcgt acatcaagca
      841 ccccgccgac atccccgact acctcaagca gtccttcccc gagggcttca agtgggagcg
      901 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
      961 cggcaccctc atctaccacg tgaagttcat cggcgtgaac ttcccctccg acggccccgt
     1021 aatgcagaag aagactctgg gctgggagcc ctccactgag cgcaactacc cccgcgacgg
     1081 cgtgctgaag ggcgagaacc acatggcgct gaagctgaag ggcggcggcc actacctgtg
     1141 tgagttcaag tccatctaca tggccaagaa gcccgtgaag ctgcccggct accactacgt
     1201 ggactacaag ctcgacatca cctcccacaa cgaggactac accgtggtgg agcagtacga
     1261 gcgcgccgag gcccgccacc acctgttcca gactcacggt atggacgaat tgtacaagca
     1321 cgacgaattg taaatccgta atcgttaatc cgcaaataac gtaaaaaccc gcttcggcgg
     1381 gtttttttat ggggggagtt tagggaaaga gcatttgtca gctggatcag cagatgggaa
     1441 ccggatactc ctcagcctcc agagtagcca atgagacgcc gagcggccgc ttatttgccg
     1501 actaccttgg tgatctcgcc tttcacgtag tggacaaatt cttccaactg atctgcgcgc
     1561 gaggccaagc gatcttcttc ttgtccaaga taagcctgtc tagcttcaag tatgacgggc
     1621 tgatactggg ccggcaggcg ctccattgcc cagtcggcag cgacatcctt cggcgcgatt
     1681 ttgccggtta ctgcgctgta ccaaatgcgg gacaacgtaa gcactacatt tcgctcatcg
     1741 ccagcccagt cgggcggcga gttccatagc gttaaggttt catttagcgc ctcaaataga
     1801 tcctgttcag gaaccggatc aaagagttcc tccgccgctg gacctaccaa ggcaacgcta
     1861 tgttctcttg cttttgtcag caagatagcc agatcaatgt cgatcgtggc tggctcgaag
     1921 atacccgcaa gaatgtcatt gcgctgccat tctccaaatt gcagttcgcg cttagctgga
     1981 taacgccacg gaatgatgtc gtcgtgcaca acaatggtga cttctacagc gcggagaatc
     2041 tcgctctctc caggggaagc cgaagtttcc aaaaggtcgt tgatcaaagc tcgccgcgtt
     2101 gtttcatcaa gccttacggt caccgtaacc agcaaatcaa tatcactgtg tggcttcagg
     2161 ccgccatcca ctgcggagcc gtacaaatgt acggccagca acgtcggttc gagatggcgc
     2221 tcgatgacgc caactacctc tgatagttga gtcgatactt cggcgatcac cgcttccctc
     2281 ataatgttta actttgtttt agggcgactg ccctgctgcg taacatcgtt gctgctccat
     2341 aacatcaaac atcgacccac ggcgtaacgc gcttgctgct tggatgcccg aggcatagac
     2401 tgtaccccaa aaaaacagtc ataacaagcc atgaaaaccg ccactgcgcc gttaccaccg
     2461 ctgcgttcgg tcaaggttct ggaccagttg cgtgagcgca tacgctactt gcattacagc
     2521 ttacgaaccg aacaggctta tgtccactgg gttcgtgcct tcatccgttt ccacggtgtg
     2581 cgtcacccgg caaccttggg tagcagcgaa gtcgaggcat ttctgtcctg gctggtcatg
     2641 accaaaatcc cttaacgtga gtcagcctgc cgccttgggc cgggtgatgt cgtacttgcc
     2701 cgccgcgaac tcggttaccg tccagcccag cgcgaccagc tccggcaacg cctcgcgcac
     2761 ccgctggcgg cgcttgcgca tggtcgaacc actggcctct gacggccaga catagccgca
     2821 caaggtatct atggaagcct tgccggtttt gccggggtcg atccagccac acagccgctg
     2881 gtgcagcagg cgggcggttt cgctgtccag cgcccgcacc tcgtccatgc tgatgcgcac
     2941 atgctggccg ccacccatga cggcctgcgc gatcaagggg ttcagggcca cgtacaggcg
     3001 cccgtccgcc tcgtcgctgg cgtactccga cagcagccga aacccctgcc gcttgcggcc
     3061 attctgggcg atgatggata ccttccaaag gcgctcgatg cagtcctgta tgtgcttgag
     3121 cgccccacca ctatcgacct ctgccccgat ttcctttgcc agcgcccgat agctaccttt
     3181 gaccaccatg gcatcagcgg tgacggcctc ccacttgggt tccaggaaca gccggagctg
     3241 ccgtccgcct tcggtcttgg gttccgggcc aagcactagg ccattaggcc cagccatggc
     3301 caccagccct tgcaggatgc gcagatcatc agcgcccagc ggctccgggc cgctgaactc
     3361 gatccgcttg ccgtcgccgt agtcatacgt cacgtccagc ttgctgcgct tgcgctcgcc
     3421 ccgcttgagg gcacggaaca ggccgggggc cagacagtgc gccgggtcgt gccggacgtg
     3481 gctgaggctg tgcttgttct taggcttcac cacggggcac ccccttgctc ttgcgctgcc
     3541 tctccagcac ggcgggcttg agcaccccgc cgtcatgccg cctgaaccac cgatcagcga
     3601 acggtgcgcc atagttggcc ttgctcacac cgaagcggac gaagaaccgg cgctggtcgt
     3661 cgtccacacc ccattcctcg gcctcggcgc tggtcatgct cgacaggtag gactgccagc
     3721 ggatgttatc gaccagtacc gagctgcccc ggctggcctg ctgctggtcg cctgcgccca
     3781 tcatggccgc gcccttgctg gcatggtgca ggaacacgat agagcacccg gtatcggcgg
     3841 cgatggcctc catgcgaccg atgacctggg ccatggggcc gctggcgttt tcttcctcga
     3901 tgtggaaccg gcgcagcgtg tccagcacca tcaggcggcg gccctcggcg gcgcgcttga
     3961 ggccgtcgaa ccactccggg gccatgatgt tgggcaggct gccgatcagc ggctggatca
     4021 gcaggccgtc agccacggct tgccgttcct cggcgctgag gtgcgcccca agggcgtgca
     4081 ggcggtgatg aatggcggtg ggcgggtctt cggcgggcag gtagatcacc gggccggtgg
     4141 gcagttcgcc cacctccagc agatccggcc cgcctgcaat ctgtgcggcc agttgcaggg
     4201 ccagcatgga tttaccggca ccaccgggcg acaccagcgc cccgaccgta ccggccacca
     4261 tgttgggcaa aacgtagtcc agcggtggcg gcgctgctgc gaacgcctcc agaatattga
     4321 taggcttatg ggtagccatt gattgcctcc tttgcaggca gttggtggtt aggcgctggc
     4381 ggggtcacta cccccgccct gcgccgctct gagttcttcc aggcactcgc gcagcgcctc
     4441 gtattcgtcg tcggtcagcc agaacttgcg ctgacgcatc cctttggcct tcatgcgctc
     4501 ggcatatcgc gcttggcgta cagcgtcagg gctggccagc aggtcgccgg tctgcttgtc
     4561 cttttggtct ttcatatcag tcaccgagaa acttgccggg gccgaaaggc ttgtcttcgc
     4621 ggaacaagga caaggtgcag ccgtcaaggt taaggctggc catatcagcg actgaaaagc
     4681 ggccagcctc ggccttgttt gacgtataac caaagccacc gggcaaccaa tagcccttgt
     4741 cacttttgat caggtagacc gaccctgaag cgcttttttc gtattccata aaaccccctt
     4801 ctgtgcgtga gtactcatag tataacaggc gtgagtacca acgcaagcac tacatgctga
     4861 aatctggccc gcccctgtcc atgcctcgct ggcggggtgc cggtgcccgt gccagctcgg
     4921 cccgcgcaag ctggacgctg ggcagaccca tgaccttgct gacggtgcgc tcgatgtaat
     4981 ccgcttcgtg gccgggcttg cgctctgcca gcgctgggct ggcctcggcc atggccttgc
     5041 cgatttcctc ggcactgcgg ccccggctgg ccagcttctg cgcggcgata aagtcgcact
     5101 tgctgaggtc atcaccgaag cgcttgacca gcccggccat ctcgctgcgg tactcgtcca
     5161 gcgccgtgcg ccggtggcgg ctaagctgcc gctcgggcag ttcgaggctg gccagcctgc
     5221 gggccttctc ctgctgccgc tgggcctgct cgatctgctg gccagcctgc tgcaccagcg
     5281 ccgggccagc ggtggcggtc ttgcccttgg attcacgcag cagcacccac ggctgataac
     5341 cggcgcgggt ggtgtgcttg tccttgcggt tggtgaagcc cgccaagcgg ccatagtggc
     5401 ggctgtcggc gctggccggg tcggcgtcgt actcgctggc cagcgtccgg gcaatctgcc
     5461 cccgaagttc accgcctgcg gcgtcggcca ccttgaccca tgcctgatag ttcttcgggc
     5521 tggtttccac taccagggca ggctcccggc cctcggcttt catgtcatcc aggtcaaact
     5581 cgctgaggtc gtccaccagc accagaccat gccgctcctg ctcggcgggc ctgatataca
     5641 cgtcattgcc ctgggcattc atccgcttga gccatggcgt gttctggagc acttcggcgg
     5701 ctgaccattc ccggttcatc atctggccgg tggtggcgtc cctgacgccg atatcgaagc
     5761 gctcacagcc catggccttg agctgtcggc ctatggcctg caaagtcctg tcgttcttca
     5821 tcgggccacc aagcgcagcc agatcgagcc gtcctcggtt gtcagtggcg tcaggtcgag
     5881 caagagcaac gatgcgatca gcagcaccac cgtaggcatc atggaagcca gcatcacggt
     5941 tagccatagc ttccagtgcc acccccgcga cgcgctccgg gcgctctgcg cggcgctgct
     6001 cacctcggcg gctacctccc gcaactcttt ggccagctcc acccatgccg cccctgtctg
     6061 gcgctgggct ttcagccact ccgccgcctg cgcctcgctg gcctgctggg tctggctcat
     6121 gacctgccgg gcttcgtcgg ccagtgtcgc catgctctgg gccagcggtt cgatctgctc
     6181 cgctaactcg ttgatgcctc tggatttctt cactctgtcg attgcgttca tggtctattg
     6241 cctcccggta ttcctgtaag tcgatgatct gggcgttggc ggtgtcgatg ttcagggcca
     6301 cgtctgcccg gtcggtgcgg atgccccggc cttccatctc caccacgttc ggccccaggt
     6361 gaacaccggg caggcgctcg atgccctgcg cctcaagtgt tctgtggtca atgcgggcgt
     6421 cgtggccagc ccgctctaat gcccggttgg catggtcggc ccatgcctcg cgggtctgct
     6481 caagccatgc cttgggcttg agcgcttcgg tcttctgtgc cccgcccttc tccggggtct
     6541 tgccgttgta ccgcttgaac cactgagcgg cgggccgctc gatgccgtca ttgatccgct
     6601 cggagatcat caggtggcag tgcgggttct cgccgccacc ggcatggatg gccagcgtat
     6661 acggcaggcg ctcggcaccg gtcaggtgct gggcgaactc ggacgccagc gccttctgct
     6721 ggtcgagggt cagctcgacc ggcagggcaa attcgacctc cttgaacagc cgcccattgg
     6781 cgcgttcata caggtcggca gcatcccagt agtcggcggg ccgctcgacg aactccggca
     6841 tgtgcccgga ttcggcgtgc aagacttcat ccatgtcgcg ggcatacttg ccttcgcgct
     6901 ggatgtagtc ggccttggcc ctggccgatt ggccgcccga cctgctgccg gttttcgccg
     6961 taaggtgata aatcgccatg ctgcctcgct gttgcttttg cttttcggct ccatgcaatg
     7021 gccctcggag agcgcaccgc ccgaagggtg gccgttaggc cagtttctcg aagagaaacc
     7081 ggtaagtgcg ccctccccta caaagtaggg tcgggattgc cgccgctgtg cctccatgat
     7141 agcctacgag acagcacatt aacaatgggg tgtcaagatg gttaagggga gcaacaaggc
     7201 ggcggatcgg ctggccaagc tcgaagaaca acgagcgcga atcaatgccg aaattcagcg
     7261 ggtgcgggca agggaacagc agcaagagcg caagaacgaa acaaggcgca aggtgctggt
     7321 gggggccatg attttggcca aggtgaacag cagcgagtgg ccggaggatc ggctcatggc
     7381 ggcaatggat gcgtaccttg aacgcgacca cgaccgcgcc ttgttcggtc tgccgccacg
     7441 ccagaaggat gagccgggct gaatgatcga ccgagacagg ccctgcgggg ctgcacacgc
     7501 gcccccaccc ttcgggtagg gggaaaggcc gctaaagcgg ctaaaagcgc tccagcgtat
     7561 ttctgcgggg tttggtgtgg ggtttagcgg gctttgcccg cctttccccc tgccgcgcag
     7621 cggtggggcg gtgtgtagcc tagcgcagcg aatagaccag ctatccggcc tctggccggg
     7681 catattgggc aagggcagca gcgccccaca agggcgctga taaccgcgcc tagtggatta
     7741 ttcttagata atcatggatg gatttttcca acaccccgcc agcccccgcc cctgctgggt
     7801 ttgcaggttt gggggcgtga cagttattgc aggggttcgt gacagttatt gcaggggggc
     7861 gtgacagtta ttgcaggggt tcgtgacagt tagtacggga gtgacgggca ctggctggca
     7921 atgtctagca acggcaggca tttcggctga gggtaaaaga actttccgct aagcgataga
     7981 ctgtatgtaa acacagtat
//
