LOCUS       sequence-260954-        8285 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RSF1010-based broad host-range plasmid with RK2 bom site expressing 
            YFP and Sp/Sm resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8285)
  AUTHORS   Bishe B, Taton A, Golden JW
  TITLE     Modification of RSF1010-Based Broad-Host-Range Plasmids for Improved
            Conjugation and Cyanobacterial Bioprospecting.
  JOURNAL   iScience. 2019 Oct 25;20:216-228. doi: 10.1016/j.isci.2019.09.002. 
            Epub 2019 Sep 10.
  PUBMED    31585408
REFERENCE   2  (bases 1 to 8285)
  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: "iScience.";
            date: "2019-10-25"; volume: "20:216-228. doi"; pages: " 
            10.1016/j.isci.2019.09.002. Epub 2019 Sep 10"
COMMENT     Sequence Label: pAM5409
FEATURES             Location/Qualifiers
     source          1..8285
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(50..77)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     protein_bind    87..111
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(join(127..840,841..843,844..846))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVRFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(172..193)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     521..540
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(join(877..883,884..899,900..905))
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     CDS             1341..2129
                     /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"
     oriT            2271..2380
                     /note="incP origin of transfer"
     terminator      2476..2519
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     rep_origin      2682..3076
                     /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            3418..3505
                     /label=RSF1010 oriT
                     /note="origin of transfer of the broad-host-range plasmid 
                     RSF1010 (Scholz et al., 1989)"
     CDS             4744..5715
                     /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
                     PWVLLRESKGKTATAGPALVQQAGQQIEQAQRQQEKARRLASLELSERQLSRHRRTALD
                     EYRSEMAGLVKRFGDDLSKCDFIAAQKLASRGRSAEEIGKAMAEASPALAERKPGHEAD
                     YIERTVSKVMGLPSVQLARAELARAPAPRQRGMDRGGPDFSM"
     CDS             6226..7065
                     /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             7052..7903
                     /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"
ORIGIN
        1 tccattgttc tagaatcccc ccgggggccc ccgattctag aaagcttcaa aaaggccatc
       61 cgtcaggatg gccttctcct gcagggccga gtttgtacaa gaaagctggg tcggcgcgcc
      121 caccctttac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc
      181 cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagggcgg actggtagct
      241 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
      301 gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt
      361 gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc
      421 cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg
      481 gttcaccagg gtgtcgccct cgaacctcac ctcggcgcgg gtcttgtagt tgccgtcgtc
      541 cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc
      601 gtgctgcttc atgtggtcgg ggtagcgggc gaagcactgc aggccgtagc cgaaggtggt
      661 cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag
      721 cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac
      781 gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct
      841 caccatctta agacctcctt tatttaaata gggtaccatt atacgagccg atgattaatt
      901 gtcaatctca attcgaaacc ggtatccggc ggcggcggcg ggataccggt ttcgaatgcg
      961 tggagaccga aaccttgcgc tcgttcgcca gccaggacag aaatgcctcg acttcgctgc
     1021 tgcccaaggt tgccgggtga cgcacaccgt ggaaacggat gaaggcacga acccagtgga
     1081 cataagcctg ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca cgcaactggt
     1141 ccagaacctt gaccgaacgc agcggtggta acggcgcagt ggcggttttc atggcttgtt
     1201 atgactgttt ttttggggta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg
     1261 ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt tacgcagcag ggcagtcgcc
     1321 ctaaaacaaa gttaaacatc atgagggaag cggtgatcgc cgaagtatcg actcaactat
     1381 cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc gtacatttgt
     1441 acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg ctggttacgg
     1501 tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt ttggaaactt
     1561 cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt gttgtgcacg
     1621 acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga gaatggcagc
     1681 gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat ctggctatct
     1741 tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg gaggaactct
     1801 ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta acgctatgga
     1861 actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg tcccgcattt
     1921 ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac tgggcaatgg
     1981 agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct tatcttggac
     2041 aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc cactacgtga
     2101 aaggcgagat caccaaggta gtcggcaagg ccggccagcc cgcctaatga gcgggctttt
     2161 ttttggcgcg ccgctagcat ccgcgcgcgc gcgcgcgatg tcgcacgata tacaggattt
     2221 tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcc gggcaggata
     2281 ggtgaagtag gcccacccgc gagcgggtgt tccttcttca ctgtccctta ttcgcacctg
     2341 gcggtgctca acgggaatcc tgctctgcga ggctggccgg ctaccgccgg cgtaacagat
     2401 gagggcaagc ggatggctga tgaaaccaag ccaaccagtc tacggtcagc gaaccaatca
     2461 gcagggtcat cgctagaaat catccttagc gaaagctaag gatttttttt atctgaatta
     2521 attctcgcga gggccgccac atgaggcatg ttccgcgtcc ttgcaatact gtgtttacat
     2581 acagtctatc gcttagcgga aagttctttt accctcagcc gaaatgcctg ccgttgctag
     2641 acattgccag ccagtgcccg tcactcccgt actaactgtc acgaacccct gcaataactg
     2701 tcacgccccc ctgcaataac tgtcacgaac ccctgcaata actgtcacgc ccccaaacct
     2761 gcaaacccag caggggcggg ggctggcggg gtgttggaaa aatccatcca tgattatcta
     2821 agaataatcc actaggcgcg gttatcagcg cccttgtggg gcgctgctgc ccttgcccaa
     2881 tatgcccggc cagaggccgg atagctggtc tattcgctgc gctaggctac acaccgcccc
     2941 accgctgcgc ggcaggggga aaggcgggca aagcccgcta aaccccacac caaaccccgc
     3001 agaaatacgc tggagcgctt ttagccgctt tagcggcctt tccccctacc cgaagggtgg
     3061 gggcgcgtgt gcagccccgc agggcctgtc tcggtcgatc attcagcccg gctcatcctt
     3121 ctggcgtggc ggcagaccga acaaggcgcg gtcgtggtcg cgttcaaggt acgcatccat
     3181 tgccgccatg agccgatcct ccggccactc gctgctgttc accttggcca aaatcatggc
     3241 ccccaccagc accttgcgcc ttgtttcgtt cttgcgctct tgctgctgtt cccttgcccg
     3301 cacccgctga atttcggcat tgattcgcgc tcgttgttct tcgagcttgg ccagccgatc
     3361 cgccgccttg ttgctcccct taaccatctt gacaccccat tgttaatgtg ctgtctcgta
     3421 ggctatcatg gaggcacagc ggcggcaatc ccgaccctac tttgtagggg agggcgcact
     3481 taccggtttc tcttcgagaa actggcctaa cggccaccct tcgggcggtg cgctctccga
     3541 gggccattgc atggagccga aaagcaaaag caacagcgag gcagcatggc gatttatcac
     3601 cttacggcga aaaccggcag caggtcgggc ggccaatcgg ccagggccaa ggccgacttc
     3661 atccagcgcg aaggcaagta tgcccgcgac atggatgaag tcttgcacgc cgaatccggg
     3721 cacatgccgg agttcgtcga gcggcccgcc gactactggg atgctgccga cctgtatgaa
     3781 cgcgccaatg ggcggctgtt caaggaggtc gaatttgccc tgccggtcga gctgaccctc
     3841 gaccagcaga aggcgctggc gtccgagttc gcccagcacc tgaccggtgc cgagcgcctg
     3901 ccgtatacgc tggccatcca tgccggtggc ggcgagaacc cgcactgcca cctgatgatc
     3961 tccgagcgga tcaatgacgg catcgagcgg cccgccgctc agtggttcaa gcggtacaac
     4021 ggcaagaccc cggagaaggg cggggcacag aagaccgaag cgctcaagcc caaggcatgg
     4081 cttgagcaga cccgcgaggc atgggccgac catgccaacc gggcattaga gcgggcaggc
     4141 cacgacgccc gcattgacca cagaacactt gaggcgcagg gcatcgagcg cctgcccggt
     4201 gttcacctgg ggccgaacgt ggtggagatg gaaggccggg gcatccgcac cgaccgggca
     4261 gacgtggccc tgaacatcga caccgccaac gcccagatca tcgacttaca ggaataccgg
     4321 gaggcaatag accatgaacg caatcgacag agtgaagaaa tccagaggca tcaacgagtt
     4381 agcggagcag atcgaaccgc tggcccagag catggcgaca ctggccgacg aagcccggca
     4441 ggtcatgagc cagacccagc aggccagcga ggcgcaggcg gcggagtggc tgaaagccca
     4501 gcgccagaca ggggcggcat gggtggagct ggccaaagag ttgcgggagg tagccgccga
     4561 ggtgagcagc gccgcgcaga gcgcccggag cgcgtcgcgg gggtggcact ggaagctatg
     4621 gctaaccgtg atgctggctt ccatgatgcc tacggtggtg ctgctgatcg catcgttgct
     4681 cttgctcgac ctgacgccac tgacaaccga ggacggctcg atctggctgc gcttggtggc
     4741 ccgatgaaga acgacaggac tttgcaggcc ataggccgac agctcaaggc catgggctgt
     4801 gagcgcttcg atatcggcgt cagggacgcc accaccggcc agatgatgaa ccgggaatgg
     4861 tcagccgccg aagtgctcca gaacacgcca tggctcaagc ggatgaatgc ccagggcaat
     4921 gacgtgtata tcaggcccgc cgagcaggag cggcatggtc tggtgctggt ggacgacctc
     4981 agcgagtttg acctggatga catgaaagcc gagggccggg agcctgccct ggtagtggaa
     5041 accagcccga agaactatca ggcatgggtc aaggtggccg acgccgcagg cggtgaactt
     5101 cgggggcaga ttgcccggac gctggccagc gagtacgacg ccgacccggc cagcgccgac
     5161 agccgccact atggccgctt ggcgggcttc accaaccgca aggacaagca caccacccgc
     5221 gccggttatc agccgtgggt gctgctgcgt gaatccaagg gcaagaccgc caccgctggc
     5281 ccggcgctgg tgcagcaggc tggccagcag atcgagcagg cccagcggca gcaggagaag
     5341 gcccgcaggc tggccagcct cgaactgtcc gagcggcagc ttagccgcca ccggcgcacg
     5401 gcgctggacg agtaccgcag cgagatggcc gggctggtca agcgcttcgg tgatgacctc
     5461 agcaagtgcg actttatcgc cgcgcagaag ctggccagcc ggggccgcag tgccgaggaa
     5521 atcggcaagg ccatggccga ggccagccca gcgctggcag agcgcaagcc cggccacgaa
     5581 gcggattaca tcgagcgcac cgtcagcaag gtcatgggtc tgcccagcgt ccagcttgcg
     5641 cgggccgagc tggcacgggc accggcaccc cgccagcgag gcatggacag gggcgggcca
     5701 gatttcagca tgtagtgctt gcgttggtac tcacgcctgt tatactatga gtactcacgc
     5761 acagaagggg gttttatgga atacgaaaaa agcgcttcag ggtcggtcta cctgatcaaa
     5821 agtgacaagg gctattggtt gcccggtggc tttggttata cgtcaaacaa ggccgaggct
     5881 ggccgctttt cagtcgctga tatggccagc cttaaccttg acggctgcac cttgtccttg
     5941 ttccgcgaag acaagccttt cggccccggc aagtttctcg gtgactgata tgaaagacca
     6001 aaaggacaag cagaccggcg acctgctggc cagccctgac gctgtacgcc aagcgcgata
     6061 tgccgagcgc atgaaggcca aagggatgcg tcagcgcaag ttctggctga ccgacgacga
     6121 atacgaggcg ctgcgcgagt gcctggaaga actcagagcg gcgcagggcg ggggtagtga
     6181 ccccgccagc gcctaaccac caactgcctg caaaggaggc aatcaatggc tacccataag
     6241 cctatcaata ttctggaggc gttcgcagca gcgccgccac cgctggacta cgttttgccc
     6301 aacatggtgg ccggtacggt cggggcgctg gtgtcgcccg gtggtgccgg taaatccatg
     6361 ctggccctgc aactggccgc acagattgca ggcgggccgg atctgctgga ggtgggcgaa
     6421 ctgcccaccg gcccggtgat ctacctgccc gccgaagacc cgcccaccgc cattcatcac
     6481 cgcctgcacg cccttggggc gcacctcagc gccgaggaac ggcaagccgt ggctgacggc
     6541 ctgctgatcc agccgctgat cggcagcctg cccaacatca tggccccgga gtggttcgac
     6601 ggcctcaagc gcgccgccga gggccgccgc ctgatggtgc tggacacgct gcgccggttc
     6661 cacatcgagg aagaaaacgc cagcggcccc atggcccagg tcatcggtcg catggaggcc
     6721 atcgccgccg ataccgggtg ctctatcgtg ttcctgcacc atgccagcaa gggcgcggcc
     6781 atgatgggcg caggcgacca gcagcaggcc agccggggca gctcggtact ggtcgataac
     6841 atccgctggc agtcctacct gtcgagcatg accagcgccg aggccgagga atggggtgtg
     6901 gacgacgacc agcgccggtt cttcgtccgc ttcggtgtga gcaaggccaa ctatggcgca
     6961 ccgttcgctg atcggtggtt caggcggcat gacggcgggg tgctcaagcc cgccgtgctg
     7021 gagaggcagc gcaagagcaa gggggtgccc cgtggtgaag cctaagaaca agcacagcct
     7081 cagccacgtc cgacacgacc cggcgcactg tctggccccc ggcctgttcc gtgccctcaa
     7141 gcggggcgag cgcaagcgca gcaagctgga cgtgacgtat gactacggcg acggcaagcg
     7201 gatcgagttc agcggcccgg agccgctggg cgctgatgat ctgcgcatcc tgcaagggct
     7261 ggtggccatg gctgggccta atggcctagt gcttggcccg gaacccaaga ccgaaggcgg
     7321 acggcagctc cggctgttcc tggaacccaa gtgggaggcc gtcaccgctg atgccatggt
     7381 ggtcaaaggt agctatcggg cgctggcaaa ggaaatcggg gcagaggtcg atagtggtgg
     7441 ggcgctcaag cacatacagg actgcatcga gcgcctttgg aaggtatcca tcatcgccca
     7501 gaatggccgc aagcggcagg ggtttcggct gctgtcggag tacgccagcg acgaggcgga
     7561 cgggcgcctg tacgtggccc tgaacccctt gatcgcgcag gccgtcatgg gtggcggcca
     7621 gcatgtgcgc atcagcatgg acgaggtgcg ggcgctggac agcgaaaccg cccgcctgct
     7681 gcaccagcgg ctgtgtggct ggatcgaccc cggcaaaacc ggcaaggctt ccatagatac
     7741 cttgtgcggc tatgtctggc cgtcagaggc cagtggttcg accatgcgca agcgccgcca
     7801 gcgggtgcgc gaggcgttgc cggagctggt cgcgctgggc tggacggtaa ccgagttcgc
     7861 ggcgggcaag tacgacatca cccggcccaa ggcggcaggc tgaccccccc cactctattg
     7921 taaacaagac atttttatct tttatattca atggcttatt ttcctgctaa ttggtaatac
     7981 catgaaaaat accatgctca gaaaaggctt aacaatattt tgaaaaattg cctactgagc
     8041 gctgccgcac agctccatag gccgctttcc tggctttgct tccagatgta tgctcttctg
     8101 ctcccgaacg ccagcaagac gtagcccagc gcgtcggcca gcttgcaatt cgcgctaact
     8161 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     8221 cgcgagaatt aattcaaaaa aagcccgctc attaagcggg ctaattcgag ggcgagacga
     8281 ggact
//
