LOCUS       sequence-410025-        5584 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses E2 Crimson on a broad host range pBBR1 origin plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5584)
  AUTHORS   Elston KM, Phillips LE, Leonard SP, Young E, Holley JC, Ahsanullah 
            T, McReynolds B, Moran NA, Barrick JE
  TITLE     The Pathfinder plasmid toolkit for genetically engineering newly 
            isolated bacteria enables the study of Drosophila-colonizing 
            Orbaceae.
  JOURNAL   ISME Commun. 2023 May 24;3(1):49. doi: 10.1038/s43705-023-00255-3.
  PUBMED    37225918
REFERENCE   2  (bases 1 to 5584)
  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"; journalName: "ISME 
            Commun."; date: "2023-05-24"; volume: "3(1):49. doi"; pages: " 
            10.1038/s43705-023-00255-3"
COMMENT     Sequence Label: pBTK1009 (pSL9)
FEATURES             Location/Qualifiers
     source          1..5584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..663
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETDEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     CDS             1481..2155
                     /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     1879..1898
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     terminator      2214..2285
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2301..2328
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     CDS             complement(2385..3200)
                     /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             complement(2385..3200)
                     /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"
     primer_bind     3108..3127
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3192..3211
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4813..5584
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
ORIGIN
        1 atggccacgc agtccagaga aatcggcatt caagccaaga acaagcccgg tcactgggtg
       61 caaacggaac gcaaagcgca tgaggcgtgg gccgggctta ttgcgaggaa acccacggcg
      121 gcaatgctgc tgcatcacct cgtggcgcag atgggccacc agaacgccgt ggtggtcagc
      181 cagaagacac tttccaagct catcggacgt tctttgcgga cggtccaata cgcagtcaag
      241 gacttggtgg ccgagcgctg gatctccgtc gtgaagctca acggccccgg caccgtgtcg
      301 gcctacgtgg tcaatgaccg cgtggcgtgg ggccagcccc gcgaccagtt gcgcctgtct
      361 gtgttcagtg ccgccgtggt ggttgatcac gacgaccagg acgaatcgct gttggggcat
      421 ggcgacctgc gccgcatccc gaccctgtat ccgggcgagc agcaactacc gaccggcccc
      481 ggcgaggagc cgcccagcca gcccggcatt ccgggcatgg aaccagacct gccagccttg
      541 accgaaacgg acgaatggga acggcgcggg cagcagcgcc tgccgatgcc cgatgagccg
      601 tgttttctgg acgatggcga gccgttggag ccgccgacac gggtcacgct gccgcgccgg
      661 tagcacttgg gttgcgcagc aacccgtaag tgcgctgttc cagactatcg gctgtagccg
      721 cctcgccgcc ctataccttg tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc
      781 cacctcgacc tgaatggaag ccggcggcac ctcgctaacg gattcaccgt ttttatcagg
      841 ctctgggagg cagaataaat gatcatatcg tcaattatta cctccacggg gagagcctga
      901 gcaaactggc ctcaggcatt tgagaagcac acggtcacac tgcttccggt agtcaataaa
      961 ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga accgacgacc
     1021 gggtcgaatt tgctttcgaa tttctgccat tcatccgctt attatcactt attcaggcgt
     1081 agcaaccagg cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc
     1141 actcatcgca gtacggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     1201 aattttaaca aaatattaac gcttacaatt tccattcgcc attcaggctg cgcaactgtt
     1261 gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctggcgaaa gggggatgtg
     1321 ctgcaaggcg attaagttgg gtaacgccag ggttttccca gtcacgacgt tgtctgacag
     1381 gccccttgag accagctttg gcagtttatt cttgacatgt agtgaggggg ctggtataat
     1441 cacatagtac tgttatacag aaacagagga gatattacat atggatagca ctgagaacgt
     1501 catcaagccc ttcatgcgct tcaaggtgca catggagggc tccgtgaacg gccacgagtt
     1561 cgagatcgag ggcgtgggcg agggcaagcc ctacgagggc acccagaccg ccaagctgca
     1621 agtgaccaag ggcggccccc tgcccttcgc ctgggacatc ctgtcccccc agttcttcta
     1681 cggctccaag gcgtacatca agcaccccgc cgacatcccc gactacctca agcagtcctt
     1741 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt
     1801 gacccaggac tcctccctgc aggacggcac cctcatctac cacgtgaagt tcatcggcgt
     1861 gaacttcccc tccgacggcc ccgtaatgca gaagaagact ctgggctggg agccctccac
     1921 tgagcgcaac tacccccgcg acggcgtgct gaagggcgag aaccacatgg cgctgaagct
     1981 gaagggcggc ggccactacc tgtgtgagtt caagtccatc tacatggcca agaagcccgt
     2041 gaagctgccc ggctaccact acgtggacta caagctcgac atcacctccc acaacgagga
     2101 ctacaccgtg gtggagcagt acgagcgcgc cgaggcccgc caccacctgt tccagactca
     2161 cggtatggac gaattgtaca agcacgacga attgtaaaag gatctccagg catcaaataa
     2221 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     2281 ctctctacta gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tataagtcgg
     2341 tctcaccgag cggccgcgtg ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg
     2401 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     2461 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     2521 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     2581 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     2641 ggcaaaagct tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     2701 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgaggcga
     2761 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     2821 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     2881 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     2941 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     3001 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     3061 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     3121 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctgga gcaagacgtt
     3181 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     3241 attgttcatg atgatatatt tttatcttgt gcaatgtaac atcagagatt ttgagacaca
     3301 acgtggcttt gttgaataaa tcgaactttt gctgagttga aggatcagtc atgaccaaaa
     3361 tcccttaacg tgagccctgc gccatcagat ccttggcggc aagaaagcca tccagtttac
     3421 tttgcagggc ttcccaacct taccagaggg cgccccagct ggcaattccg gttcgcttgc
     3481 tgtccataaa accgcccagt ctagctatcg ccatgtaagc ccactgcaag ctacctgctt
     3541 tctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat tcatcccagg
     3601 tggcactttt cggggaaatg tgcgcgcccg cgttcctgct ggcgctgggc ctgtttctgg
     3661 cgctggactt cccgctgttc cgtcagcagc ttttcgccca cggccttgat gatcgcggcg
     3721 gccttggcct gcatatcccg attcaacggc cccagggcgt ccagaacggg cttcaggcgc
     3781 tcccgaaggt ctcgggccgt ctcttgggct tgatcggcct tcttgcgcat ctcacgcgct
     3841 cctgcggcgg cctgtagggc aggctcatac ccctgccgaa ccgcttttgt cagccggttg
     3901 gccacggctt ccggcgtctc aacgcgcttt gagattccca gcttttcggc caatccctgc
     3961 ggtgcatagg cgcgtggctc gaccgcttgc gggctgatgg tgacgtggcc cactggtggc
     4021 cgctccaggg cctcgtagaa cgcctgaatg cgcgtgtgac gtgccttgct gccctcgatg
     4081 ccccgttgca gccctagatc ggccacagcg gccgcaaacg tggtctggtc gcgggtcatc
     4141 tgcgctttgt tgccgatgaa ctccttggcc gacagcctgc cgtcctgcgt cagcggcacc
     4201 acgaacgcgg tcatgtgcgg gctggtttcg tcacggtgga tgctggccgt cacgatgcga
     4261 tccgccccgt acttgtccgc cagccacttg tgcgccttct cgaagaacgc cgcctgctgt
     4321 tcttggctgg ccgacttcca ccattccggg ctggccgtca tgacgtactc gaccgccaac
     4381 acagcgtcct tgcgccgctt ctctggcagc aactcgcgca gtcggcccat cgcttcatcg
     4441 gtgctgctgg ccgcccagtg ctcgttctct ggcgtcctgc tggcgtcagc gttgggcgtc
     4501 tcgcgctcgc ggtaggcgtg cttgagactg gccgccacgt tgcccatttt cgccagcttc
     4561 ttgcatcgca tgatcgcgta tgccgccatg cctgcccctc ccttttggtg tccaaccggc
     4621 tcgacggggg cagcgcaagg cggtgcctcc ggcgggccac tcaatgcttg agtatactca
     4681 ctagactttg cttcgcaaag tcgtgaccgc ctacggcggc tgcggcgccc tacgggcttg
     4741 ctctccgggc ttcgccctgc gcggtcgctg cgctcccttg ccagcccgtg gatatgtgga
     4801 cgatggccgc gagcggccac cggctggctc gcttcgctcg gcccgtggac aaccctgctg
     4861 gacaagctga tggacaggct gcgcctgccc acgagcttga ccacagggat tgcccaccgg
     4921 ctacccagcc ttcgaccaca tacccaccgg ctccaactgc gcggcctgcg gccttgcccc
     4981 atcaattttt ttaattttct ctggggaaaa gcctccggcc tgcggcctgc gcgcttcgct
     5041 tgccggttgg acaccaagtg gaaggcgggt caaggctcgc gcagcgaccg cgcagcggct
     5101 tggccttgac gcgcctggaa cgacccaagc ctatgcgagt gggggcagtc gaagggcgaa
     5161 gcccgcccgc ctgccccccg agcctcacgg cggcgagtgc gggggttcca agggggcagc
     5221 gccaccttgg gcaaggccga aggccgcgca gtcgatcaac aagccccgga ggggccactt
     5281 tttgccggag ggggagccgc gccgaaggcg tgggggaacc ccgcaggggt gcccttcttt
     5341 gggcaccaaa gaactagata tagggcgaaa tgcgaaagac ttaaaaatca acaacttaaa
     5401 aaaggggggt acgcaacagc tcattgcggc accccccgca atagctcatt gcgtaggtta
     5461 aagaaaatct gtaattgact gccactttta cgcaacgcat aattgttgtc gcgctgccga
     5521 aaagttgcag ctgattgcgc atggtgccgc aaccgtgcgg cacccctacc gcatggagat
     5581 aagc
//
