LOCUS Exported 8116 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses Lambda Red recombinases and a dominant negative MutL allele all controlled by temperature sensitve cI857 repressor for high precision and efficiency MAGE experiments. Cam resistance marker.. ACCESSION . VERSION . KEYWORDS pORTMAGE-4 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8116) AUTHORS Nyerges A, Csorgo B, Nagy I, Balint B, Bihari P, Lazar V, Apjok G, Umenhoffer K, Bogos B, Posfai G, Pal C TITLE A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. JOURNAL Proc Natl Acad Sci U S A. 2016 Feb 16. pii: 201520040. PUBMED 26884157 REFERENCE 2 (bases 1 to 8116) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8116 /organism="synthetic DNA construct" /mol_type="other DNA" misc_feature 4..22 /label=Tn5 ME /note="hyperactive mosaic end for Tn5 transposase recognition (Reznikoff et al., 2004)" CDS complement(2014..2694) /codon_start=1 /gene="exo" /product="5' to 3' double-stranded DNA exonuclease in the lambda Red system" /label=Exo /note="creates 3' single-stranded DNA tails" /translation="MTPDIILQRTGIDVRAVEQGEDAWHKLRLGVITASEVHNVIAKPR SGKKWPDMKMSYFHTLLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESP IIYRDESMRTACSPDGLCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMW VTRKNAWYFANYDPRMKREGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFG EQWR" CDS complement(2691..3476) /codon_start=1 /gene="bet" /product="single-stranded DNA binding recombinase in the lambda Red system" /label=Beta /note="mediates pairing of a single-stranded DNA with a complementary target" /translation="MSTALATLAGKLAERVGMDSVDPQELITTLRQTAFKGDASDAQFI ALLIVANQYGLNPWTKEIYAFPDKQNGIVPVVGVDGWSRIINENQQFDGMDFEQDNESC TCRIYRKDRNHPICVTEWMDECRREPFKTREGREITGPWQSHPKRMLRHKAMIQCARLA FGFAGIYDKDEAERIVENTAYTAERQPERDITPVNDETMQEINTLLIALDKTWDDDLLP LCSQIFRRDIRASSELTQAEAVKALGFLKQKAAEQKVAA" protein_bind 3909..3923 /label=boxB /bound_moiety="lambda-N peptide" /note="lambda N peptide binding site consisting of an RNA hairpin within the N-utliization (nut) site of the lambda bacteriophage transcript (Tan and Frankel, 1995)" CDS complement(4523..5182) /codon_start=1 /gene="cat" /product="chloramphenicol acetyltransferase" /label=CmR /note="confers resistance to chloramphenicol" /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL KTVKKNKHKFYPAFIHILARLMNAHPKFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA" primer_bind 5097..5116 /label=CAT-R /note="Chloramphenicol resistance gene, reverse primer" promoter complement(5183..5285) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" promoter complement(5368..5439) /gene="bla" /label=AmpR promoter CDS complement(5551..6264) /codon_start=1 /gene="cIts" /product="temperature-sensitive variant of the phage lambda repressor" /label=-lambda repressor (ts) /note="thermosensitivity is conferred by the A67T mutation" /translation="MSTKKKPLTQEQLEDARRLKAIYEKKKNELGLSQESVADKMGMGQ SGVGALFNGINALNAYNAALLTKILKVSVEEFSPSIAREIYEMYEAVSMQPSLRSEYEY PVFSHVQAGMFSPELRTFTKGDAERWVSTTKKASDSAFWLEVEGNSMTAPTGSKPSFPD GMLILVDPEQAVEPGDFCIARLGGDEFTFKKLIRDSGQVFLQPLNPQYPMIPCNESCSV VGKVIASQWPEETFG" RBS 6353..6361 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" misc_feature 6376..6394 /label=Tn5 ME /note="hyperactive mosaic end for Tn5 transposase recognition (Reznikoff et al., 2004)" rep_origin 6621..7392 /label=pBBR1 oriV /note="replication origin of the broad-host-range plasmid pBBR1 from Bordetella bronchiseptica; requires the pBBR1 Rep protein for replication" CDS 7393..8055 /codon_start=1 /product="replication protein for the broad-host-range plasmid pBBR1 from Bordetella bronchiseptica" /label=pBBR1 Rep /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR" ORIGIN 1 cagctgtctc ttatacacat ctccgctgtg ctttcagtgg atttcggata acagaaaggc 61 cgggaaatac ccagcctcgc tttgtaacgg agtagacgaa agtgattgcg cctacccgga 121 tattatcgtg aggatgctca tcgccatttc actcatcttt cagggctttt atcgccggat 181 gtaaatcaac agattgtaac agaccacccg gcggcgtttt cacaagttgc ggacataacc 241 gttccacgtc cgccagcagg gttatggcct gtgccattga ccactgcgca tgttcgctca 301 tcagatttcg tgcaatccac tgcgcaatat tgccaggttc gaatacggac tgcttcgcca 361 ggtagcctat cagttcagga atcaagattt gtaaattttg ttggcgtaag ggtaaaggca 421 ctgccctgat ggtcacatgc tgtgcatctg actggaaatc aatacccaat tccgccaggg 481 cagactgcgc tttttctaat gccgattttt cttcggcaga aacttttagc cgcaacggaa 541 tcagcagcgg ctgggcgcaa acgggcgctt cacccggcgt caattgtgcc tgacgcagcc 601 aacgttctgc cactggcaag gataaaagtg aaatgttgcc gtcgcgctcc agcaacgcac 661 agtcggaatg gacgatagtc agtacccgac caaaactctg actgttcgcc gcaagtgcag 721 gttcctgcgg ttccggcgct tttaattttt gcatcggcgc gggcgtttgc aaaagctggc 781 gatacacttc accttgctgt ttctggtagc ctggctgcgc attcggccag ggggcagccg 841 gacgactgcc tgatgctggc gcaggagtgt agcgcggagc taccggctca cgagctgccg 901 gttctgcaaa gtgattgcgc cccgccgcca cgcggttttc cggaatggaa cgcggtgcag 961 gttggggttc atcgtccagc ggtagcggcg tttccagttg ctgttgtagc acgctcagca 1021 cgccctgata gataaaatca tgcaccagac gcgactgatg gaaacgcact tcgtgtttgg 1081 cggggtgcac gttgacgtcc acctgatgtg ggtcgatctc cagatacaac acaaatgccg 1141 gttgctgatc ggcccccagt ttgtcttcgc aggcctggcg gatcgcgtga ttgatcaggc 1201 gatcgcgcat catgcgaccg ttcacgtagc aatactgaat ttctgccagt gcgggcgtgg 1261 tgtgatttgg atcggccacc cagccgcgta gcgtgagatc gccgtgttgc cattcaatcg 1321 ccagcgcttg ttcaagaaaa gcggtgccgc aaatcgcgcc taagcgccgt tctttttgcc 1381 cgccttccgg cactgcgcgg tactgacgca caattttacc gttatgcgac aggttgatcg 1441 tgacgtcgaa acgcgccagc gcaatgcggc ggatgatctc atcaatgtgg ttaaattcgg 1501 ttttctcggt gcgcaggaat ttgcgccgcg ccggggtgtt gtagaacaga tccagcacct 1561 ccagcgtcgt ccccacagga tgcgccgccg gttttaccgt cacgttcata tcgcgccctt 1621 cggcataggc ctgccaggct tcctgctgtt ctgcggtgcg tgaagtgagc gtcaggcggg 1681 aaaccgaact gatactcgcc agcgcctcac cgcgaaagcc caggctgata atggcttcga 1741 gatcgtccag agaggcgatt ttactggtgg catgacgagc cagcgccagc gccagctcat 1801 cttttttgat accgcagccg ttatcacgaa tgcggataag tttcgcccca ccgcgttcga 1861 tatcaatatc gatacgcgtc gcacctgcat cgaggctgtt tttcactagt tctttgacta 1921 ccgacgcagg tcgctcgacc acctcacctg cggcaatctg gttcgccagt tgtggcggta 1981 agacctgaat tggcattttg taatcctcct ctctcatcgc cattgctccc caaatacaaa 2041 accaatttca gccagtgcct cgtccatttt ttcgatgaac tccggcacga tctcgtcaaa 2101 actcgccatg tacttttcat cccgctcaat cacgacataa tgcaggcctt cacgcttcat 2161 acgcgggtca tagttggcaa agtaccaggc attttttcgc gtcacccaca tgctgtactg 2221 cacctgggcc atgtaagctg actttatggc ctcgaaacca ccgagccgga acttcatgaa 2281 atcccgggag gtaaacgggc atttcagttc aaggccgttg ccgtcactgc ataaaccatc 2341 gggagagcag gcggtacgca tactttcgtc gcgatagatg atcggggatt cagtaacatt 2401 cacgccggaa gtaaattcaa acagggttct ggcgtcgttc tcgtactgtt ttccccaggc 2461 cagtgcttta gcgttaactt ccggagccac accggtgcaa acctcagcaa gcagggtgtg 2521 gaagtaggac attttcatgt caggccactt ctttccggag cggggttttg ctatcacgtt 2581 gtgaacttct gaagcggtga tgacgccgag ccgtaatttg tgccacgcat cttccccctg 2641 ttcgacagct ctcacatcga tcccggtacg ctgcaggata atgtccggtg tcatgctgcc 2701 accttctgct ctgcggcttt ctgtttcagg aatccaagag cttttactgc ttcggcctgt 2761 gtcagttctg acgatgcacg aatgtcgcgg cgaaatatct gggaacagag cggcaataag 2821 tcgtcatccc atgttttatc cagggcgatc agcagagtgt taatctcctg catggtttca 2881 tcgttaaccg gagtgatgtc gcgttccggc tgacgttctg cagtgtatgc agtattttcg 2941 acaatgcgct cggcttcatc cttgtcatag ataccagcaa atccgaaggc cagacgggca 3001 cactgaatca tggctttatg acgtaacatc cgtttgggat gcgactgcca cggccccgtg 3061 atttctctgc cttcgcgagt tttgaatggt tcgcggcggc attcatccat ccattcggta 3121 acgcagatcg gatgattacg gtccttgcgg taaatccggc atgtacagga ttcattgtcc 3181 tgctcaaagt ccatgccatc aaactgctgg ttttcattga tgatgcggga ccagccatca 3241 acgcccacca ccggaacgat gccattctgc ttatcaggaa aggcgtaaat ttctttcgtc 3301 cacggattaa ggccgtactg gttggcaacg atcagtaatg cgatgaactg cgcatcgctg 3361 gcatcacctt taaatgccgt ctggcgaaga gtggtgatca gttcctgtgg gtcgacagaa 3421 tccatgccga cacgttcagc cagcttccca gccagcgttg cgagtgcagt actcattcgt 3481 tttatacctc tgaatcaata tcaacctggt ggtgagcaat ggtttcaacc atgtaccgga 3541 tgtgttctgc catgcgctcc tgaaactcaa catcgtcatc aaacgcacgg gtaatggatt 3601 ttttgctggc cccgtggcgt tgcaaatgat cgatgcatag cgattcaaac aggtgctggg 3661 gcaggccttt ttccatgtcg tctgccagtt ctgcctcttt ctcttcacgg gcgagctgct 3721 ggtagtgacg cgcccagctc tgagcctcaa gacgatcctg aatgtaataa gcgttcatgg 3781 ctgaactcct gaaatagctg tgaaaatatc gcccgcgaaa tgccgggctg attagtaatc 3841 cggaatcgca cttacggcca atgcttcgtt tcgtatcaca caccccaaag ccttctgctt 3901 tgaatgctgc ccttcttcag ggcttaattt ttaagagcgt caccttcatg gtggtcagtg 3961 cgtcctgctg atgtgctcag tatcaccgcc agtggtattt atgtcaacac cgccagagat 4021 aatttatcac cgcagatggt tatctgtatg ttttttatat gaatttattt tttgcagggg 4081 ggcattgttt ggtaggtgag agatctgaat tgctatgttt agtgagttgt atctatttat 4141 ttttcaataa atacaattgg ttatgtgttt tgggggcgat cgtgaggcaa agaaaacccg 4201 gcgctgaggc cgggttaaga gttggtagct cttgatccgg caaacaaacc accgctggta 4261 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 4321 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 4381 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 4441 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttag gatccgaggc 4501 accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag tactgttgta 4561 attcattaag cattctgccg acatggaagc catcacaaac ggcatgatga acctgaatcg 4621 ccagcggcat cagcaccttg tcgccttgcg tataatattt gcccatggtg aaaacggggg 4681 cgaagaagtt gtccatattg gccacgttta aatcaaaact ggtgaaactc acccagggat 4741 tggctgagac gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac 4801 cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt 4861 cactccagag cgatgaaaac gtttcagttt gctcatggaa aacggtgtaa caagggtgaa 4921 cactatccca tatcaccagc tcaccgtctt tcattgccat acggaatttc ggatgagcat 4981 tcatcaggcg ggcaagaatg tgaataaagg ccggataaaa cttgtgctta tttttcttta 5041 cggtctttaa aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa 5101 ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca acggtggtat 5161 atccagtgat ttttttctcc attttagctt ccttagctcc tgaaaatctc gataactcaa 5221 aaaatacgcc cggtagtgat cttatttcat tatggtgaaa gttggaacct cttacgtgcc 5281 gatcaacgtc tcattttcgc caaaagttgg cccagggctt cccggtatca acagggacac 5341 caggatttat ttattctgcg cggtaccact cttccttttt caatattatt gaagcattta 5401 tcagggttat tgtctcatga gcggatacat atttgaatga attcaaccat ttactatgtt 5461 atgttctgag gggagtgaaa attcccctaa ttcgatgaag attcttgctc aattgttatc 5521 agctatgcgc cgaccagaac accttgccga tcagccaaac gtctcttcag gccactgact 5581 agcgataact ttccccacaa cggaacaact ctcattgcat gggatcattg ggtactgtgg 5641 gtttagtggt tgtaaaaaca cctgaccgct atccctgatc agtttcttga aggtaaactc 5701 atcaccccca agtctggcta tgcagaaatc acctggctca acagcctgct cagggtcaac 5761 gagaattaac attccgtcag gaaagcttgg cttggagcct gttggtgcgg tcatggaatt 5821 accttcaacc tcaagccaga atgcagaatc actggctttt ttggttgtgc ttacccatct 5881 ctccgcatca cctttggtaa aggttctaag ctcaggtgag aacatccctg cctgaacatg 5941 agaaaaaaca gggtactcat actcacttct aagtgacggc tgcatactaa ccgcttcata 6001 catctcgtag atttctctgg cgattgaagg gctaaattct tcaacgctaa ctttgagaat 6061 ttttgtaagc aatgcggcgt tataagcatt taatgcattg atgccattaa ataaagcacc 6121 aacgcctgac tgccccatcc ccatcttgtc tgcgacagat tcctgggata agccaagttc 6181 atttttcttt ttttcataaa ttgctttaag gcgacgtgcg tcctcaagct gctcttgtgt 6241 taatggtttc ttttttgtgc tcatacgtta aatctatcac cgcaagggat aaatatctaa 6301 caccgtgcgt gttgactatt ttacctctgg cggtgataat ggttgcatgt actaaggagg 6361 ttgtatggaa caacgagatg tgtataagag acagctggcc tgcccctccc ttttggtgtc 6421 caaccggctc gacgggggca gcgcaaggcg gtgcctccgg cgggccactc aatgcttgag 6481 tatactcact agactttgct tcgcaaagtc gtgaccgcct acggcggctg cggcgcccta 6541 cgggcttgct ctccgggctt cgccctgcgc ggtcgctgcg ctcccttgcc agcccgtgga 6601 tatgtggacg atggccgcga gcggccaccg gctggctcgc ttcgctcggc ccgtggacaa 6661 ccctgctgga caagctgatg gacaggctgc gcctgcccac gagcttgacc acagggattg 6721 cccaccggct acccagcctt cgaccacata cccaccggct ccaactgcgc ggcctgcggc 6781 cttgccccat caattttttt aattttctct ggggaaaagc ctccggcctg cggcctgcgc 6841 gcttcgcttg ccggttggac accaagtgga aggcgggtca aggctcgcgc agcgaccgcg 6901 cagcggcttg gccttgacgc gcctggaacg acccaagcct atgcgagtgg gggcagtcga 6961 agggcgaagc ccgcccgcct gccccccgag cctcacggcg gcgagtgcgg gggttccaag 7021 ggggcagcgc caccttgggc aaggccgaag gccgcgcagt cgatcaacaa gccccggagg 7081 ggccactttt tgccggaggg ggagccgcgc cgaaggcgtg ggggaacccc gcaggggtgc 7141 ccttctttgg gcaccaaaga actagatata gggcgaaatg cgaaagactt aaaaatcaac 7201 aacttaaaaa aggggggtac gcaacagctc attgcggcac cccccgcaat agctcattgc 7261 gtaggttaaa gaaaatctgt aattgactgc cacttttacg caacgcataa ttgttgtcgc 7321 gctgccgaaa agttgcagct gattgcgcat ggtgccgcaa ccgtgcggca cccctaccgc 7381 atggagataa gcatggccac gcagtccaga gaaatcggca ttcaagccaa gaacaagccc 7441 ggtcactggg tgcaaacgga acgcaaagcg catgaggcgt gggccgggct tattgcgagg 7501 aaacccacgg cggcaatgct gctgcatcac ctcgtggcgc agatgggcca ccagaacgcc 7561 gtggtggtca gccagaagac actttccaag ctcatcggac gttctttgcg gacggtccaa 7621 tacgcagtca aggacttggt ggccgagcgc tggatctccg tcgtgaagct caacggcccc 7681 ggcaccgtgt cggcctacgt ggtcaatgac cgcgtggcgt ggggccagcc ccgcgaccag 7741 ttgcgcctgt cggtgttcag tgccgccgtg gtggttgatc acgacgacca ggacgaatcg 7801 ctgttggggc atggcgacct gcgccgcatc ccgaccctgt atccgggcga gcagcaacta 7861 ccgaccggcc ccggcgagga gccgcccagc cagcccggca ttccgggcat ggaaccagac 7921 ctgccagcct tgaccgaaac ggaggaatgg gaacggcgcg ggcagcagcg cctgccgatg 7981 cccgatgagc cgtgttttct ggacgatggc gagccgttgg agccgccgac acgggtcacg 8041 ctgccgcgcc ggtagcactt gggttgcgca gcaacccgta agtgcgctgt tccagactat 8101 cggctgtacc gcctcg //