LOCUS Exported 3302 bp ds-DNA circular SYN 17-JUN-2022 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pKD4 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3302) AUTHORS Datsenko KA, Wanner BL TITLE One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. JOURNAL Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5. PUBMED 10829079 REFERENCE 2 (bases 1 to 3302) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 17, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3302 /organism="synthetic DNA construct" /mol_type="other DNA" promoter complement(82..186) /gene="bla" /label=AmpR promoter terminator 276..362 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 454..481 /label=rrnB T2 terminator /note="transcription terminator T2 from the E. coli rrnB gene" protein_bind complement(559..606) /label=FRT /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="FLP-mediated recombination occurs in the 8-bp core sequence TCTAGAAA (Turan and Bode, 2011)." CDS 966..1760 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(1020..1039) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 1630..1649 /label=Neo-F /note="Neomycin resistance gene, forward primer" protein_bind complement(1951..1984) /label=FRT (minimal) /bound_moiety="FLP recombinase from the Saccharomyces cerevisiae 2u plasmid" /note="supports FLP-mediated excision but not integration (Turan and Bode, 2011)" rep_origin 2033..2421 /label=R6K gamma ori /note="gamma replication origin from E. coli plasmid R6K; requires the R6K initiator protein pi for replication" CDS complement(2523..81) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind 3146..3165 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 61 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 121 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 181 tccgcgcaca tttccccgaa aagtgccacc tgcatcgatg gcccccgatg gtagtgtggg 241 gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaatgaaag gctcagtcga 301 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa 361 atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac 421 gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt 481 ttgcgtggcc agtgccaagc ttgcatgcag attgcagcat tacacgtctt gagcgattgt 541 gtaggctgga gctgcttcga agttcctata ctttctagag aataggaact tcggaatagg 601 aacttcaaga tcccccacgc tgccgcaagc actcagggcg caagggctgc taaaggaagc 661 ggaacacgta gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact 721 gggctatctg gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc 781 ttacatggcg atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag 841 ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc 901 cgccaaggat ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt 961 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc 1021 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc 1081 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg 1141 aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag 1201 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg 1261 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg 1321 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac 1381 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg 1441 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc 1501 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg 1561 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc 1621 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc 1681 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc 1741 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc 1801 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg 1861 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt 1921 cttcgcccac cccagcttca aaagcgctct gaagttccta tactttctag agaataggaa 1981 cttcggaata ggaactaagg aggatattca tatggaccat ggctaattcc catgtcagcc 2041 gttaagtgtt cctgtgtcac tcaaaattgc tttgagaggc tctaagggct tctcagtgcg 2101 ttacatccct ggcttgttgt ccacaaccgt taaaccttaa aagctttaaa agccttatat 2161 attctttttt ttcttataaa acttaaaacc ttagaggcta tttaagttgc tgatttatat 2221 taattttatt gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacgttag 2281 ccatgagagc ttagtacgtt agccatgagg gtttagttcg ttaaacatga gagcttagta 2341 cgttaaacat gagagcttag tacgtgaaac atgagagctt agtacgtact atcaacaggt 2401 tgaactgctg atcttcagat cctctacgcc ggacgcatcg tggccggatc ttgcggccgc 2461 aaaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 2521 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 2581 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 2641 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 2701 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 2761 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 2821 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 2881 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 2941 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 3001 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 3061 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 3121 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 3181 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 3241 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 3301 ag //