LOCUS Exported 8285 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION RSF1010-based broad host-range plasmid with RK2 bom site expressing YFP and Sp/Sm resistance. ACCESSION . VERSION . KEYWORDS pAM5409 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8285 /organism="synthetic DNA construct" /mol_type="other DNA" terminator 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(127..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" primer_bind 780..801 /label=EGFP-N /note="EGFP, reverse primer" promoter complement(877..905) /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" CDS 1341..2126 /codon_start=1 /gene="aadA" /product="aminoglycoside adenylyltransferase (Murphy, 1985)" /label=SmR /note="confers resistance to spectinomycin and streptomycin" /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVG" 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" 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 //