LOCUS Exported 5756 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION IncP-based replicating vector expressing dTomato under tac promoter.. ACCESSION . VERSION . KEYWORDS pAWP89 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5756) AUTHORS Puri AW, Owen S, Chu F, Chavkin T, Beck DA, Kalyuzhnaya MG, Lidstrom ME TITLE Genetic tools for the industrially promising methanotroph Methylomicrobium buryatense. JOURNAL Appl Environ Microbiol. 2014 Dec 29. pii: AEM.03795-14. PUBMED 25548049 REFERENCE 2 (bases 1 to 5756) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5756 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 11..642 /label=oriV /note="incP origin of replication" rep_origin 951..1539 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1440..1459 /label=pBR322ori-F /note="pBR322 origin, forward primer" promoter 1676..1704 /label=tac promoter /note="strong E. coli promoter; hybrid between the trp and lac UV5 promoters" CDS 1733..2434 /codon_start=1 /product="dimeric variant of DsRed fluorescent protein (Shaner et al., 2004)" /label=dTomato /note="mammalian codon-optimized" /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH HLFLYGMDELYK" primer_bind complement(1866..1884) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2134..2153 /label=mCherry-F /note="mCherry, forward primer" primer_bind 2328..2351 /label=DsRed1-C /note="DsRed1, forward primer" CDS complement(2562..3377) /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 3285..3304 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 3369..3388 /label=pENTR-R /note="pENTR vectors, reverse primer" CDS complement(3681..4829) /codon_start=1 /product="trans-acting replication protein that binds to and activates oriV" /label=trfA /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR" CDS complement(5101..5472) /codon_start=1 /gene="traJ" /product="oriT-recognizing protein" /label=traJ /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSL*AYLLAV GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE KQDELGKVMMGVVRPRAEP" oriT 5505..5614 /note="incP origin of transfer" ORIGIN 1 gaccctttcc gacgctcacc gggctggttg ccctcgccgc tgggctggcg gccgtctatg 61 gccctgcaaa cgcgccagaa acgccgtcga agccgtgtgc gagacaccgc ggccgccggc 121 gttgtggata cctcgcggaa aacttggccc tcactgacag atgaggggcg gacgttgaca 181 cttgaggggc cgactcaccc ggcgcggcgt tgacagatga ggggcaggct cgatttcggc 241 cggcgacgtg gagctggcca gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt 301 tcccacagat gatgtggaca agcctgggga taagtgccct gcggtattga cacttgaggg 361 gcgcgactac tgacagatga ggggcgcgat ccttgacact tgaggggcag agtgctgaca 421 gatgaggggc gcacctattg acatttgagg ggctgtccac aggcagaaaa tccagcattt 481 gcaagggttt ccgcccgttt ttcggccacc gctaacctgt cttttaacct gcttttaaac 541 caatatttat aaaccttgtt tttaaccagg gctgcgccct gtgcgcgtga ccgcgcacgc 601 cgaagggggg tgccccccct tctcgaaccc tcccggcccg ctaacgcggg cctcccatcc 661 ccccaggggc tgcgcccctc ggccgcgaac ggcctcaccc caaaaatggc agccaagctg 721 acccgctagg gacgtgaagt cgattccttc gatggttagc aatcaaagac tcagagtgct 781 gtctggagcg tgaatctaac ggtacgtatc tcgattgctc ggtcgctatt cgcactctgc 841 gaaagttcgt accgctcatt cactaggttg cgaatcatga ccaaaatccc ttaacgtgag 901 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 961 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 1021 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 1081 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 1141 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 1201 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 1261 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 1321 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 1381 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 1441 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 1501 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 1561 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 1621 gattctgtgg ataaccgtat taccgccttt gagtgagctg ctctgaaatg agctgttgac 1681 aattaatcat cggctcgtat aatgtgtgga ggtattcaca caggaaacag ctatggtgag 1741 caagggcgag gaggtcatca aagagttcat gcgcttcaag gtgcgcatgg agggctccat 1801 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca 1861 gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc 1921 cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca tccccgatta 1981 caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg 2041 cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga tctacaaggt 2101 gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga agaccatggg 2161 ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca 2221 ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga ccatctacat 2281 ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc tggacatcac 2341 ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg gccgccacca 2401 cctgttcctg tacggcatgg acgagctgta caagtagttg tcgggaagat gcgtgatctg 2461 atccttccac tcagcaaaag ttcgatttat tcaacaaagc cgccgtcccg tcaagtcagc 2521 gtaatgctct gccagtgtta caaccaatta accaattctg attagaaaaa ctcatcgagc 2581 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc 2641 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg 2701 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca 2761 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc 2821 aaaagcttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca 2881 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat 2941 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac 3001 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat 3061 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa 3121 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct 3181 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc 3241 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta 3301 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctcgagca agacgtttcc 3361 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt 3421 gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg 3481 tggctttcga cttccggcaa gctatacgcg ccctagaatt gtcaatttta atcctctgtt 3541 tatcggcagt tcgtagagcg cgccgtgcgt cccgagcgat actgagcgaa gcaagtgcgt 3601 cgagcagtgc ccgcttgttc ctgaaatgcc agtaaagcgc tggctgctga acccccagcc 3661 ggaactgacc ccacaaggcc ctagcgtttg caatgcacca ggtcatcatt gacccaggcg 3721 tgttccacca ggccgctgcc tcgcaactct tcgcaggctt cgccgacctg ctcgcgccac 3781 ttcttcacgc gggtggaatc cgatccgcac atgaggcgga aggtttccag cttgagcggg 3841 tacggctccc ggtgcgagct gaaatagtcg aacatccgtc gggccgtcgg cgacagcttg 3901 cggtacttct cccatatgaa tttcgtgtag tggtcgccag caaacagcac gacgatttcc 3961 tcgtcgatca ggacctggca acgggacgtt ttcttgccac ggtccaggac gcggaagcgg 4021 tgcagcagcg acaccgattc caggtgccca acgcggtcgg acgtgaagcc catcgccgtc 4081 gcctgtaggc gcgacaggca ttcctcggcc ttcgtgtaat accggccatt gatcgaccag 4141 cccaggtcct ggcaaagctc gtagaacgtg aaggtgatcg gctcgccgat aggggtgcgc 4201 ttcgcgtact ccaacacctg ctgccacacc agttcgtcat cgtcggcccg cagctcgacg 4261 ccggtgtagg tgatcttcac gtccttgttg acgtggaaaa tgaccttgtt ttgcagcgcc 4321 tcgcgcggga ttttcttgtt gcgcgtggtg aacagggcag agcgggccgt gtcgtttggc 4381 atcgctcgca tcgtgtccgg ccacggcgca atatcgaaca aggaaagctg catttccttg 4441 atctgctgct tcgtgtgttt cagcaacgcg gcctgcttgg cctcgctgac ctgttttgcc 4501 aggtcctcgc cggcggtttt tcgcttcttg gtcgtcatag ttcctcgcgt gtcgatggtc 4561 atcgacttcg ccaaacctgc cgcctcctgt tcgagacgac gcgaacgctc cacggcggcc 4621 gatggcgcgg gcagggcagg gggagccagt tgcacgctgt cgcgctcgat cttggccgta 4681 gcttgctgga ccatcgagcc gacggactgg aaggtttcgc ggggcgcacg catgacggtg 4741 cggcttgcga tggtttcggc atcctcggcg gaaaaccccg cgtcgatcag ttcttgcctg 4801 tatgccttcc ggtcaaacgt ccgattcatt caccctcctt gcgggattgc cccgactcac 4861 gccggggcaa tgtgccctta ttcctgattt gacccgcctg gtgccttggt gtccagataa 4921 tccaccttat cggcaatgaa gtcggtcccg tagaccgtct ggccgtcctt ctcgtacttg 4981 gtattccgaa tcttgccctg cacgaatacc agctccgcga agtcgctctt cttgatggag 5041 cgcatgggga cgtgcttggc aatcacgcgc accccccggc cgttttagcg gctaaaaaag 5101 tcatggctct gccctcgggc ggaccacgcc catcatgacc ttgccaagct cgtcctgctt 5161 ctcttcgatc ttcgccagca gggcgaggat cgtggcatca ccgaaccgcg ccgtgcgcgg 5221 gtcgtcggtg agccagagtt tcagcaggcc gcccaggcgg cccaggtcgc cattgatgcg 5281 ggccagctcg cggacgtgct catagtccac gacgcccgtg attttgtagc cctggccgac 5341 ggccagcagg taggcctaca ggctcatgcc ggccgccgcc gccttttcct caatcgctct 5401 tcgttcgtct ggaaggcagt acaccttgat aggtgggctg cccttcctgg ttggcttggt 5461 ttcatcagcc atccgcttgc cctcatctgt tacgccggcg gtagccggcc agcctcgcag 5521 agcaggattc ccgttgagca ccgccaggtg cgaataaggg acagtgaaga aggaacaccc 5581 gctcgcgggt gggcctactt cacctatcct gcccggctga cgccgttgga tacaccaagg 5641 aaagtctaca cgaacccttt ggcaaaatcc tgtatatcgt gcgaaaaagg atggatatac 5701 cgaaaaaatc gctataatga ccccgaagca gggttatgca gcggaaaaga tccgtc //