LOCUS Exported 5782 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Recombination-mediated cassette exchange with target genomic loci. ACCESSION . VERSION . KEYWORDS pLibacceptorV2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5782) AUTHORS Urtecho G, Tripp AD, Insigne K, Kim H, Kosuri S TITLE Systematic Dissection of Sequence Elements Controlling sigma70 Promoters Using a Genomically-Encoded Multiplexed Reporter Assay in E. coli. JOURNAL Biochemistry. 2018 Feb 1. doi: 10.1021/acs.biochem.7b01069. PUBMED 29388765 REFERENCE 2 (bases 1 to 5782) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5782 /organism="synthetic DNA construct" /mol_type="other DNA" protein_bind 600..633 /label=lox66 /bound_moiety="Cre recombinase" /note="Right element (RE) mutant of loxP (Araki et al., 2010). Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." CDS 1025..1819 /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 GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(1079..1098) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 1689..1708 /label=Neo-F /note="Neomycin resistance gene, forward primer" CDS complement(2166..3116) /codon_start=1 /gene="rep101 encoding an A56V mutation" /product="temperature-sensitive version of the RepA protein needed for replication with the pSC101 origin (Armstrong et al., 1984)" /label=Rep101(Ts) /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI" rep_origin 3164..3386 /label=pSC101 ori /note="low-copy replication origin that requires the Rep101 protein" CDS 5179..428 /codon_start=1 /product="site-specific recombinase" /label=Cre /note="Cre is a site-specific recombinase from bacteriophage P1. Recombination occurs at loxP sequences." /translation="MANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE DGD" ORIGIN 1 cgctggttag caccgcaggt gtagagaagg cacttagcct gggggtaact aaactggtcg 61 agcgatggat ttccgtctct ggtgtagctg atgatccgaa taactacctg ttttgccggg 121 tcagaaaaaa tggtgttgcc gcgccatctg ccaccagcca gctatcaact cgcgccctgg 181 aagggatttt tgaagcaact catcgattga tttacggcgc taaggatgac tctggtcaga 241 gatacctggc ctggtctgga cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg 301 ctggagtttc aataccggag atcatgcaag ctggtggctg gaccaatgta aatattgtca 361 tgaactatat ccgtaacctg gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg 421 gcgattaact agtggggaag cttatcgatg ataagctgtc aaacatgaga attacaactt 481 atatcgtatg gggctgactt caggtgctac atttgaagag ataaattgca ctgaaatcta 541 gaaatatttt atctgattcc tcatcagtgc caacatagta agccagtaga attccgcgta 601 taacttcgta tagcatacat tatacgaacg gtagacgaac aataaggcct cccaaatcgg 661 ggggcctttt ttattgataa caaaactcgg taccaaattc cagaaaagag acgctgaaaa 721 gcgtcttttt tcgttttggt ccagtgccat gttatccctg aagtcgacct agctcgatcg 781 atggctatcc ctgcaggcat cgggtgggat ttagctacag gtatgcgtag gagtcaagtc 841 agtttcacct gttttacgta aaaacccgct tcggcgggtt tttacttttg gtccggcaat 901 taaaaaagcg gctaaccacg ccgctttttt tacgtctgca aaaaagagta ttgacttcgc 961 atctttttgt acccataatt atttcattac tagaaagatc ttttaagaag gagatatata 1021 ctagatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 1081 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 1141 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 1201 actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 1261 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 1321 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 1381 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 1441 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 1501 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc 1561 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 1621 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 1681 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 1741 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 1801 tcttgacgag ttcttctgat gatactagag atcgagaagg acacggttaa tactaggcct 1861 gctggctggt aatcgccagc aggccttttt atttggggga gagggaagtc atgaaaaaac 1921 taacctttga aattcgatct ccaccacatc agctctgaag caacgtaaaa aaacccgccc 1981 cggcgggttt ttttataccc gtagtatccc cacttatcta caatagctgt taccgttcgt 2041 atatggtttc ttatacgaag ttatatatgg acagttttcc ctttgatatg taacggtgaa 2101 cagttgttct acttttgttt gttagtcttg atgcttcact gatagataca agagccataa 2161 gaacctcaga tccttccgta tttagccagt atgttctcta gtgtggttcg ttgtttttgc 2221 gtgagccatg agaacgaacc attgagatca tacttacttt gcatgtcact caaaaatttt 2281 gcctcaaaac tggtgagctg aatttttgca gttaaagcat cgtgtagtgt ttttcttagt 2341 ccgttacgta ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc attcattttt 2401 atctggttgt tctcaagttc ggttacgaga tccatttgtc tatctagttc aacttggaaa 2461 atcaacgtat cagtcgggcg gcctcgctta tcaaccacca atttcatatt gctgtaagtg 2521 tttaaatctt tacttattgg tttcaaaacc cattggttaa gccttttaaa ctcatggtag 2581 ttattttcaa gcattaacat gaacttaaat tcatcaaggc taatctctat atttgccttg 2641 tgagttttct tttgtgttag ttcttttaat aaccactcat aaatcctcat agagtatttg 2701 ttttcaaaag acttaacatg ttccagatta tattttatga atttttttaa ctggaaaaga 2761 taaggcaata tctcttcact aaaaactaat tctaattttt cgcttgagaa cttggcatag 2821 tttgtccact ggaaaatctc aaagccttta accaaaggat tcctgatttc cacagttctc 2881 gtcatcagct ctctggttgc tttagctaat acaccataag cattttccct actgatgttc 2941 atcatctgaa cgtattggtt ataagtgaac gataccgtcc gttctttcct tgtagggttt 3001 tcaatcgtgg ggttgagtag tgccacacag cataaaatta gcttggtttc atgctccgtt 3061 aagtcatagc gactaatcgc tagttcattt gctttgaaaa caactaattc agacatacat 3121 ctcaattggt ctaggtgatt ttaatcacta taccaattga gatgggctag tcaatgataa 3181 ttactagtcc ttttcctttg agttgtgggt atctgtaaat tctgctagac ctttgctgga 3241 aaacttgtaa attctgctag accctctgta aattccgcta gacctttgtg tgtttttttt 3301 gtttatattc aagtggttat aatttataga ataaagaaag aataaaaaaa gataaaaaga 3361 atagatccca gccctgtgta taactcacta ctttagtcag ttccgcagta ttacaaaagg 3421 atgtcgcaaa cgctgtttgc tcctctacaa aacagacctt aaaaccctaa aggcttaagt 3481 agcaccctcg caagctcggt tgcggccgca atcgggcaaa tcgctgaata ttccttttgt 3541 ctccgaccat caggcacctg agtcgctgtc tttttcgtga cattcagttc gctgcgctca 3601 cggctctggc agtgaatggg ggtaaatggc actacaggcg ccttttatgg attcatgcaa 3661 ggaaactacc cataatacaa gaaaagcccg tcacgggctt ctcagggcgt tttatggcgg 3721 gtctgctatg tggtgctatc tgactttttg ctgttcagca gttcctgccc tctgattttc 3781 cagtctgacc acttcggatt atcccgtgac aggtcattca gactggctaa tgcacccagt 3841 aaggcagcgg tatcatcaac tttttttaag gcagttattg gtgcccttaa acgcctggtt 3901 gctacgcctg aataagtgat aataagcgga tgaatggcag aaattcgaat agttacggct 3961 tatgacatct ttgtggacac atcattcact ttttattcac atccggccct gaactcgcta 4021 ggacttgccc cggtgcattt tttaaatacc cgcgaaaaat agagctgatc gtcaaatcca 4081 acattgcgcc caacggtcgc tatcggcatt cgcgtagtgc taagcagaag tttcgcctgg 4141 ctgatacgct gatcttcgcg ccagctcaat acgctaatgc ctaactgctg gcggaacaga 4201 tgtgataacc gggagggcga caggcagaca tgctgggcga cgctggcgat atcaaaatgg 4261 ctgtccgcca gatggtcgct gatatactgg caggcatcgc gcacacggct atccatcggc 4321 gggtgcaacg actcattaat taccgccata cgtctgagca acaactgctc cagcagattg 4381 atcgccagta gctcagaata gcgaccttcc ccttgcccgg cgctgatgat ctgcccgaac 4441 agttcgctga aatgcggctg gcgcgcctcg tccgggcgga aaaatcctgt ctgggcaaag 4501 attgtcggcc aggtcagcca ctcctgccag taggcgcgag gccggaaata aacccactgg 4561 tgataccact cgctggcgtc cggatgccgt ccatagtgat gaatctcgcc cggcggaaac 4621 aataatatat cgccaggccg acagacaaac tgctcgccat tattattaat gacgccctct 4681 ccgcggatgg tcaggttaag aatatatccc ttcatgccca acggacgatc gataaaaaaa 4741 tccagatatc cattcgcttc gatcggcgtc agcccggcga ccagatgggc attaaatgaa 4801 tatcccggca atagcggatc attttgcgtt tcagccatga tttctctacc ccccgatgtt 4861 cagagaagaa acaaattgtc catatcgacc aggacgacag agcttccgtc tccgcaagac 4921 tttgcgcttg atgaaagcac gtatcaaccc cgcttgtgaa aagcgctttg taacaaaagc 4981 gtacagttca ggcgataaaa ttaagtaaca gaagtgtcta taactatggc tggaatgtcc 5041 acattgaata tttgcacagc gtcacacttt gcaaagcatt agcatttttg tccataagat 5101 tagcggatcc tgcctgacgg tttttgccgc gactctctat aatttctcca tacctgtttt 5161 tctggatgga gtaagaccat ggccaattta ctgaccgtac accaaaattt gcctgcatta 5221 ccggtcgatg caacgagtga tgaggttcgc aagaacctga tggacatgtt cagggatcgc 5281 caggcgtttt ctgagcatac ctggaaaatg cttctgtccg tttgccggtc gtgggcggca 5341 tggtgcaagt tgaataaccg gaaatggttt cccgcagaac ctgaagatgt tcgcgattat 5401 cttctatatc ttcaggcgcg cggtctggca gtaaaaacta tccagcaaca tttgggccag 5461 ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa gtgacagcaa tgctgtttca 5521 ctggttatgc ggcggatccg aaaagaaaac gttgatgccg gtgaacgtgc aaaacaggct 5581 ctagcgttcg aacgcactga tttcgaccag gttcgttcac tcatggaaaa tagcgatcgc 5641 tgccaggata tacgtaatct ggcatttctg gggattgctt ataacaccct gttacgtata 5701 gccgaaattg ccaggatcag ggttaaagat atctcacgta ctgacggtgg gagaatgtta 5761 atccatattg gcagaacgaa aa //