LOCUS Exported 9435 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Input plasmid: expresses three serine recombinases downstream of three inducible promoters.. ACCESSION . VERSION . KEYWORDS pNR220 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 9435) AUTHORS Roquet N, Soleimany AP, Ferris AC, Aaronson S, Lu TK TITLE Synthetic recombinase-based state machines in living cells. JOURNAL Science. 2016 Jul 22;353(6297):aad8559. doi: 10.1126/science.aad8559. Epub 2016 Jul 21. PUBMED 27463678 REFERENCE 2 (bases 1 to 9435) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..9435 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(156..744) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(236..255) /label=pBR322ori-F /note="pBR322 origin, forward primer" terminator 832..926 /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(957..1751) /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(1068..1087) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 1678..1697 /label=Neo-R /note="Neomycin resistance gene, reverse primer" misc_feature 1906..1927 /label=BioBrick prefix /note="BioBrick prefix for parts that do not start with ""ATG""" terminator 1962..2048 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4492..4563 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" protein_bind 4772..4790 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 4797..4815 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" terminator 6388..6415 /label=T7Te terminator /note="phage T7 early transcription terminator" CDS complement(6498..7376) /codon_start=1 /gene="araC" /product="L-arabinose regulatory protein" /label=araC /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWAYFRPRAYW HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE EKVNDVAVKLS" promoter 7403..7687 /gene="araBAD" /label=araBAD promoter /note="promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction (Guzman et al., 1995)" primer_bind 7610..7629 /label=pBAD Forward /note="For vectors with E. coli araBAD promoter, forward primer" CDS 7741..7758 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 9226..9258 /codon_start=1 /product="C-terminal peptide that mediates degradation in bacteria through the ClpXP and ClpAP proteases (McGinness et al., 2006)" /label=ssrA tag /note="wild-type LAA variant" /translation="AAKDENYALAA" misc_feature 9262..9282 /label=BioBrick suffix /note="universal suffix for all parts" terminator 9283..9340 /label=his operon terminator /note="This putative transcriptin terminator from the E. coli his operon has a 2-bp deletion introduced during synthesis. Its efficiency has not been determined." primer_bind complement(9364..9381) /label=L4440 /note="L4440 vector, forward primer" ORIGIN 1 cctagctagg gcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg 61 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag 121 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac 181 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga 241 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt 301 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc 361 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc 421 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta 481 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat 541 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca 601 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct 661 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt 721 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct 781 cagtggaacg aaaactcacg ttaagggatt ttggtcatga ctagtgcttg gattctcacc 841 aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga gttctgaggt 901 cattactgga tctatcaaca ggagtccaag cgagctctcg aaccccagag tcccgctcag 961 aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg 1021 taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta 1081 gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc gatgaatcca 1141 gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg 1201 agatcctcgc cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc 1261 ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt 1321 gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta 1381 tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat 1441 gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg 1501 acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct 1561 gcctcgtcct gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg 1621 cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc 1681 cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct 1741 tgttcaatca tgcgaaacga tcctcatcct gtctcttgat cagatcttga tcccctgcgc 1801 catcagatcc ttggcggcaa gaaagccatc cagtttactt tgcagggctt cccaacctta 1861 ccagagggcg ccccagctgg caattccgac gtcaaagggc ccagagaatt cgcggccgct 1921 tctagagcag caaaacccgt accctaggtc tagggcggcg gatttgtcct actcaggaga 1981 gcgttcaccg acaaacaaca gataaaacga aaggcccagt ctttcgactg agcctttcgt 2041 tttatttgat gcctctagca cgcgtaccat gggaaccccc gggctgcagc tagagccatt 2101 caatagtaac ttgttcacca tcaatataaa ttttgtttat tagtgatttt aaataaagtt 2161 gcttttctct gaactctaaa gagtcaaaat caactgttgc taaatcagct aaattttctt 2221 gtatcttttt gtttttcttc aattcttcgt tagcttctat ttgtgattca taataattaa 2281 tttgagcatc aatatcattc atcatagaat caagttctga aacttcatac gagccattta 2341 tatataaatc aaataatcgt tttttctttg catgttctat tttaagcttt tcatttaagc 2401 tatctaattc atcttcttta tctacatttc tggaagcgaa actataatta ttcacacgat 2461 taataattaa ttcttcaagt ttgtcagctc tccaaatttt attcccgcat ttttcgagtt 2521 catgagtatg tttataagtc ttgcaactat aatatctata atgatatttt ttaccacgcg 2581 acattgtatc ttttctacga tgaacaaagc ctaacccgca tttactacaa actactaaat 2641 tatttagcaa cgatgctgaa tctctattca tgttcggatt tttacccata cgagtaaata 2701 tttcttgaac tctatagaat tgctcttcac tgatgatagg ttcatgaata ccttttacat 2761 gaactttatc tttatatgaa acataaccac aatacaaatc attagttagc cagttgttat 2821 agcgattata tgttctaact ttaaagccta atttttttag tcttttctgt aaaaaagtta 2881 tactttgttc ttcttcgaaa atatcataaa tcagttgtaa ctgttttgct tcttcttcat 2941 taatgtataa ttttgtatct ataacatcat agccgaacgt tctacctttc gcagttgtta 3001 acggaagacc tgcttcaata cgcttaattt tccccatcac catacgatct cggattgttt 3061 cgcgctctag ctgtgcgaat actgataata taccaatcat tgcacgaccg aaaggggaac 3121 tagtatcaag cgtttcagac aaactaacaa actctacatt gttttttaag aagtattctt 3181 caataagcgt tattgtgtct ctttgtgagc gggatagtct gtctaatcga tatacgacta 3241 cagcatcaat ttcgtgtagt ttacttagca tttcatttaa tgcgggacga ttcatatttg 3301 agccggagta tccgccgtca atgaaaatat cgtatacgtc ccagtccttc gagcggcaca 3361 atcctgttag tttttcagtt tgagcttgta ttgaataatt ttctacttgc tcttgagtag 3421 aaacacgtat ataaatagct gccttcatta cttatatcga cgtatttcgg gatggctaag 3481 taaccttaac gatacggtac gtttcgtatc atgtcaattg gtaacgaatc agagcgcaat 3541 aaaaaagccc ccggaaggtg atcttccggg ggctttctca tgcgttaagg gctatagcaa 3601 ttccgacggc acagctaaca ccacgtcgtc cctatctgct gccctaggtc tatgagtggt 3661 tgctggataa ctttacgggc atgcataagg ctcgtataat atattcaggg agaccacaac 3721 ggtttccctc tacaaataat tttgtttaac tttctgaatc aatcggcggc gaagggagaa 3781 atactagatg gcacgtaccc cgagccgtag cagcattggt agcctgcgta gtccgcatac 3841 ccataaagca attctgacca gcaccattga aatcctgaaa gaatgtggtt atagcggtct 3901 gagcattgaa agcgttgcac gtcgtgccgg tgcaagcaaa ccgaccattt atcgttggtg 3961 gaccaataaa gcagcactga ttgccgaagt gtatgaaaat gaaagcgaac aggtgcgtaa 4021 atttccggat ctgggtagct ttaaagccga tctggatttt ctgctgcgta atctgtggaa 4081 agtttggcgt gaaaccattt gtggtgaagc atttcgttgt gttattgcag aagcacagct 4141 ggaccctgca accctgaccc agctgaaaga tcagtttatg gaacgtcgtc gtgagatgcc 4201 gaaaaaactg gttgaaaatg ccattagcaa tggtgaactg ccgaaagata ccaatcgtga 4261 actgctgctg gatatgattt ttggtttttg ttggtatcgc ctactgaccg aacagctgac 4321 cgttgaacag gatattgaag aatttacctt cctgctgatt aatggtgttt gtccgggtac 4381 acagcgttaa ggaaacacag aaaaaagccc gcacctgaca gtgcgggctt tttttttcga 4441 ccaaaggtac tcgacgtaca tacagacctc ccactcacac tccccaggca tcaaataaaa 4501 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct 4561 ctcgggccca ctcgcagctt aagttcagcc aaaaaactta agaccgccgg tcttgtccac 4621 taccttgcag taatgcggtg gacaggatcg gcggttttct tttctcttct caacccgaat 4681 gggagtgaac accaacgcat gagaaagccc ccggaagatc accttccggg ggctttttta 4741 ttgcgcgcat tgagctaccc ggtggctaga gtccctatca gtgatagaga ttgacatccc 4801 tatcagtgat agagatactg agcacaagct tgcggctttc acacatagga gctagcgtga 4861 gagccctggt agtcatccgc ctgtcccgcg tcaccgatgc tacgacttca ccggagcgtc 4921 agctggagtc ttgccagcag ctctgcgccc agcgcggctg ggacgtcgtc ggggtagcgg 4981 aggatctgga cgtctccggg gcggtcgatc cgttcgaccg gaagcgcaga ccgaacctgg 5041 cccggtggct agcgttcgag gagcaaccgt ttgacgtgat cgtggcgtac cgggtagatc 5101 ggttgacccg atcgatccgg catcttcagc agctggtcca ctgggccgag gaccacaaga 5161 agctggtcgt ctccgcgacc gaagcgcact tcgatacgac gacgccgttt gcggcggtcg 5221 tcatcgcgct tatgggaacg gtggcgcaga tggaattaga agcgatcaaa gagcggaacc 5281 gttcggctgc gcatttcaat atccgcgccg ggaaataccg aggatccctg ccgccgtggg 5341 gatacctgcc tacgcgcgtg gacggggagt ggcggctggt gccggaccct gtgcagcgag 5401 agcgcatcct cgaggtgtat caccgcgtcg tcgacaacca cgagccgctg catctggtgg 5461 cccacgacct gaaccggcgt ggtgtcctgt cgccgaagga ctacttcgcg cagctgcaag 5521 gccgcgagcc gcagggccgg gagtggtcgg ctaccgcgct gaagcgatcg atgatctccg 5581 gggcgatgct cgggtacgcg actctgaacg gtaagaccgt ccgagacgac gacggagccc 5641 cgctggtgcg ggctgagccg atcctgaccc gtgagcagct ggaggcgctg cgcgccgagc 5701 tcgtgaagac ctcccgggcg aagcccgcgg tgtctacccc gtcgctgctg ctgcgggtgt 5761 tgttctgcgc ggtgtgcggg gagcccgcgt acaagttcgc cgggggagga cgtaagcacc 5821 cgcgctaccg ctgccgctcg atggggttcc cgaagcactg cgggaacggc acggtggcga 5881 tggccgagtg ggacgcgttc tgcgaggagc aggtactgga tctgctcggg gacgcggagc 5941 gtctggagaa agtctgggta gcgggctcgg actccgcggt cgaactcgcg gaggtgaacg 6001 cggagctggt ggacctgacg tcgctgatcg gctccccggc ctaccgggcg ggctctccgc 6061 agcgagaagc actggatgcc cgtattgcgg cgctggccgc gcggcaagag gagctggagg 6121 gcctggaggc tcgcccgtct ggctgggagt ggcgcgagac cgggcagcgg ttcggggact 6181 ggtggcggga gcaggacacc gcggcaaaga acacctggct tcggtcgatg aacgttcggc 6241 tgacgttcga cgtccgcggc gggctgactc gcacgatcga cttcggggat cttcaggagt 6301 acgagcagca tctcaggctc ggcagcgtgg tcgaacggct acacaccggg atgtcgtaat 6361 aatactagat gcattaataa tcacactggc tcaccttcgg gtgggccttt ctgcgtttat 6421 atactagaga gagaatataa aaagccagat tattaatccg gcttttttat tatttcctgc 6481 agggcggccg ccatatgtta tgacaacttg acggctacat cattcacttt ttcttcacaa 6541 ccggcacgga actcgctcgg gctggccccg gtgcattttt taaatacccg cgagaaatag 6601 agttgatcgt caaaaccaac attgcgaccg acggtggcga taggcatccg ggtggtgctc 6661 aaaagcagct tcgcctggct gatacgttgg tcctcgcgcc agcttaagac gctaatccct 6721 aactgctggc ggaaaagatg tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg 6781 ctggcgatat caaaattgct gtctgccagg tgatcgctga tgtactgaca agcctcgcgt 6841 acccgattat ccatcggtgg atggagcgac tcgttaatcg cttccatgcg ccgcagtaac 6901 aattgctcaa gcagatttat cgccagcagc tccgaatagc gcccttcccc ttgcccggcg 6961 ttaatgattt gcccaaacag gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag 7021 aaccccgtat tggcaaatat tgacggccag ttaagccatt catgccagta ggcgcgcgga 7081 cgaaagtaag cccactggtg ataccattcg cgagcctccg gatgacgacc gtagtgatga 7141 atctctcctg gcgggaacag caaaatatca cccggtcggc aaacaaattc tcgtccctga 7201 tttttcacca ccccctgacc gcgaatggtg agattgagaa tataaccttt cattcccagc 7261 ggtcggtcga taaaaaaatc gagataaccg ttggcctcaa tcggcgttaa acccgccacc 7321 agatgggcat taaacgagta tcccggcagc aggggatcat tttgcgcttc agccatactt 7381 ttcatactcc cgccattcag agaagaaacc aattgtccat attgcatcag acattgccgt 7441 cactgcgtct tttactggct cttctcgcta accaaaccgg taaccccgct tattaaaagc 7501 attctgtaac aaagcgggac caaagccatg acaaaaacgc gtaacaaaag tgtctataat 7561 cacggcagaa aagtccacat tgattatttg cacggcgtca ctctttgcta tgccatagca 7621 tttttatcca taagattagc ggatcctacc tgacgctttt tatcgcaact ctctactgtt 7681 tctccatacc gtttttttgg gctagctact agagtcacac aggaaaccta ctagatgaaa 7741 catcatcacc atcaccacca ggccggcact aagaaagtag caatctatac acgagtatcc 7801 actactaacc aagcagagga aggcttctca attgatgagc aaattgaccg tttaacaaaa 7861 tatgctgaag caatggggtg gcaagtatct gatacttata ctgatgctgg tttttcaggg 7921 gccaaacttg aacgcccagc aatgcaaaga ttaatcaacg atatcgagaa taaagctttt 7981 gatacagttc ttgtatataa gctagaccgc ctttcacgta gtgtaagaga tactctttat 8041 cttgttaagg atgtgttcac aaaaaataaa atagacttta tctcgcttaa tgaaagtatt 8101 gatacttctt ctgctatggg tagcttgttt ctcactattc tttctgcaat taatgagttt 8161 gaaagagaga atataaaaga acgcatgact atgggtaaac tagggcgagc gaaatctggt 8221 aagtctatga tgtggactaa gacagctttt gggtattacc acaacagaaa gacaggtata 8281 ttagaaattg ttcctttaca agctacaata gttgaacaaa tattcactga ttatttatca 8341 ggaatatcac ttacaaaatt aagagataaa ctcaatgaat ctggacacat cggtaaagat 8401 ataccgtggt cttatcgtac cctaagacaa acacttgata atccagttta ctgtggttat 8461 atcaaattta aggacagcct atttgaaggt atgcacaaac caattatccc ttatgagact 8521 tatttaaaag ttcaaaaaga gctagaagaa agacaacagc agacttatga aagaaataac 8581 aaccctagac ctttccaagc taaatatatg ctgtcaggga tggcaaggtg cggttactgt 8641 ggagcacctt taaaaattgt tcttggccac aaaagaaaag atggaagccg cactatgaaa 8701 tatcactgtg caaatagatt tcctcgaaaa acaaaaggaa ttacagtata taatgacaat 8761 aaaaagtgtg attcaggaac ttatgattta agtaatttag aaaatactgt tattgacaac 8821 ctgattggat ttcaagaaaa taatgactcc ttattgaaaa ttatcaatgg caacaaccaa 8881 cctattcttg atacttcgtc atttaaaaag caaatttcac agatcgataa aaaaatacaa 8941 aagaactctg atttgtacct aaatgatttt atcactatgg atgagttgaa agatcgtact 9001 gattcccttc aggctgagaa aaagctgctt aaagctaaga ttagcgaaaa taaatttaat 9061 gactctactg atgtttttga gttagttaaa actcagttgg gctcaattcc gattaatgaa 9121 ctatcatatg ataataaaaa gaaaatcgtc aacaaccttg tatcaaaggt tgatgttact 9181 gctgataatg tagatatcat atttaaattc caactcgcta ccggtgctgc taaggacgaa 9241 aactacgctc tggctgctta atactagtag cggccgctgc agtccggcaa aaaagggcaa 9301 ggtgtcacca ccctgccctt tttctttaaa accgaaaaga ttacttcgcg ttatgcaggc 9361 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 9421 ctcaaaggcg gtaat //