LOCUS 40924_615 5883 bp DNA circular SYN 17-DEC-2018 DEFINITION Cloning vector DH6-56, complete sequence. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5883) AUTHORS Huang DC, Holtz WJ, Maharbiz MM. TITLE A genetic bistable switch utilizing nonlinear protein degradation JOURNAL J Biol Eng 6 (1), 9 (2012) PUBMED 22776405 REFERENCE 2 (bases 1 to 5883) AUTHORS Huang DC, Holtz WJ, Maharbiz MM. TITLE Direct Submission JOURNAL Submitted (10-JUN-2012) Electrical Engineering and Computer Sciences, University of California, Berkeley, 656 Sutardja Dai Hall, Berkeley, CA 94720, USA REFERENCE 3 (bases 1 to 5883) TITLE Direct Submission REFERENCE 4 (bases 1 to 5883) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "J Biol Eng"; date: "2012"; volume: "6"; issue: "1"; pages: "9" COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted (10-JUN-2012) Electrical Engineering and Computer Sciences, University of California, Berkeley, 656 Sutardja Dai Hall, Berkeley, CA 94720, USA" COMMENT SGRef: number: 3; type: "Journal Article" COMMENT ##Assembly-Data-START## Assembly Method :: GENtle v. 1.9.4 Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END## FEATURES Location/Qualifiers source 1..5883 /mol_type="other DNA" /organism="synthetic DNA construct" protein_bind complement(15..36) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS complement(52..1131) /label=lacI /note="lac repressor" promoter complement(1132..1209) /label=lacI promoter protein_bind 1502..1518 /label=lac operator /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." protein_bind 1525..1541 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." 5'UTR 1569..1602 gene 1603..4002 /gene="mfLon" /label=mfLon CDS 1603..3999 /codon_start=1 /gene="mfLon" /product="mf-Lon with ssrA degrdation tag LVA" /label=mfLon /protein_id="AFN88256.1" /translation="MSKKIKLPIFQIRGSFIVPGIKENLEVGRKNTLASVNYAIKNSNN QMIAIPQIDASVEKPEFSDLHEFGILIDFEVIKEWKDNSLTISTNPIQRCKVISFFENE DQVPYAEVELIESINDFSDEELKELIEKISDAIKTKASLVTKQIKQLISGESDDLSLAF DSIMFKLAPSKILTNPEYITSPSLKTRWSIIEKIIFAEDGIITRNAESIDAARQKNEIE QELNHKLKEKMDKQQKEYYLREKMRIIKDELEDEDDSDDSSLEKYKERLAKEPFPEEVK RKIMASIKRVEALQSGTPEWNTEKNYIDWMMSIPWWEETEDLTDLKYAKKILDKHHYGM KKVKERIIEYLAVKTKTKSLKAPIITLVGPPGVGKTSLAKSIAEAVGKNFVKVSLGGVK DESEIRGHRKTYVGSMPGRIIQTMKRAKVKNPLFLLDEIDKMASDHRGDPASAMLEVLD PEQNKEFSDHYIEEPYDLSQVMFIATANYPEDIPEALYDRMEIINLSSYTEIEKVKIAQ DYLVPKAIEQHELTSEEISFTEGAINEIIKYYTREAGVRQLERHINSIIRKYIVKNLNG EMDKIVIDEKQVNDLLGKRIFDHTEKQEESQIGVVTGLAYTQFGGDILPIEVSLYPGKG NLILTGKLGEVMKESATIALTYVKSNFEKFGVDKKVFEENDIHVHVPEGAVPKDGPSAG ITITTALISALSDKPVSKEIGMTGEITLRGNVLPIGGLREKSISASRSGLKTIIIPKKN ERDLDEIPDEVKAKLKIIPAEKYEEVFAIVFKTKAANDENYALVA" misc_feature 3964..3996 /gene="mfLon" /label=degradation tag /note="degradation tag" CDS 3964..3996 /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 (LVA) /note="mutant LVA variant that confers accelerated degradation under some conditions (Andersen et al., 1998)" /translation="AANDENYALVA" terminator 4035..4106 /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" terminator 4122..4149 /label=T7Te terminator /note="phage T7 early transcription terminator" rep_origin complement(4307..4895) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" terminator complement(4983..5077) /label=lambda t0 terminator /note="transcription terminator from phage lambda" CDS complement(5101..5757) /label=CmR /note="chloramphenicol acetyltransferase" promoter complement(5758..5860) /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" ORIGIN 1 gacgtcggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct 61 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 121 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag 181 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg 241 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc 301 ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt 361 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac 421 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc 481 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag 541 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc 601 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact 661 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc 721 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg 781 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat 841 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg 901 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc 961 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac 1021 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg 1081 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac 1141 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc 1201 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca 1261 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg 1321 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca 1381 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag 1441 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga 1501 attgtgagcg gataacaatt gacattgtga gcggataaca agatactgag cacgaattca 1561 aaagatctta atatcatcga ccggccggct acgaggataa ggatgagtaa aaaaataaaa 1621 ctgcctattt tccaaatacg cggttcattt attgttcctg gaattaaaga gaatttagaa 1681 gttggaagaa aaaacacatt agcgagcgtt aattatgcga ttaaaaattc aaataatcaa 1741 atgatcgcaa taccgcagat tgatgcatct gttgaaaaac ctgaattttc agatttacat 1801 gaatttggaa ttttaattga ttttgaagta atcaaagaat ggaaagataa ttcattaact 1861 attagcacaa accctattca acgatgtaaa gtaattagct tttttgaaaa tgaagatcaa 1921 gtgccgtatg cggaagttga attaattgaa tcaattaatg attttagtga tgaagagtta 1981 aaagaattaa ttgaaaaaat ctctgatgct atcaaaacta aagctagttt agtaacaaaa 2041 caaattaaac agttaatatc aggtgaatct gatgatttga gcttagcttt tgattcaatt 2101 atgtttaaac tggctccgtc aaaaatattg acaaatccgg aatacataac atctccgtca 2161 ttaaaaacac gatggtcaat tattgaaaaa ataatttttg ctgaagatgg aatcataaca 2221 agaaatgcag aatcaattga tgcagcacga caaaagaatg aaattgaaca agaactgaac 2281 cataaattaa aagaaaaaat ggataaacaa caaaaagaat attatctgcg agaaaaaatg 2341 cgaattatca aagatgaatt agaagatgaa gatgattctg atgacagttc attagaaaaa 2401 tataaagaac gtttagctaa agaacctttc cctgaagaag tcaaacgaaa aattatggct 2461 tctatcaaac gagttgaagc acttcaatca ggaactcctg aatggaatac tgagaaaaat 2521 tacattgatt ggatgatgag tattccctgg tgggaagaaa ctgaagactt aactgattta 2581 aaatatgcta aaaaaatttt agacaaacat cattatggta tgaaaaaagt taaagaaaga 2641 attattgagt acttagctgt taaaactaaa acaaaatcac taaaagcacc aattattaca 2701 ttagttggtc ccccaggtgt aggtaaaaca agtttagcaa aatcaattgc agaagcagtt 2761 ggaaaaaact ttgttaaagt aagtttaggt ggagttaaag atgaatcaga aattcgtgga 2821 catagaaaaa cttatgttgg ttcaatgcca ggaagaatta ttcaaacaat gaaaagagca 2881 aaagtaaaaa acccattatt cttgcttgat gaaattgata aaatggcatc tgatcacaga 2941 ggtgatccag caagtgcaat gttagaggtt cttgacccag aacaaaacaa agagttttca 3001 gatcattata ttgaagaacc atatgatcta agccaagtta tgtttatagc aactgctaac 3061 tatcctgaag atattcctga agccttatat gaccgtatgg aaatcataaa tttatctagc 3121 tatactgaaa ttgaaaaagt taagatagca caagattatt tagttccaaa agcaatagaa 3181 caacatgaat taacttcaga agaaatatca tttactgaag gtgcaattaa tgaaattatt 3241 aaatattaca ctagagaagc tggtgtgcgt caattagaaa gacatattaa ttctattatt 3301 agaaagtata ttgttaaaaa tctaaatggt gaaatggata aaattgttat tgatgaaaaa 3361 caagtaaatg atttattagg taaaagaatt tttgatcaca ctgaaaaaca agaagaatct 3421 caaattggtg tggttacagg tttagcatat acacaatttg gtggagatat tttaccaatt 3481 gaagtaagtt tataccctgg taaaggaaat ttaattctta caggtaaact tggtgaagtt 3541 atgaaagaat ctgcaacaat tgcgttaact tatgttaaat caaactttga aaaatttgga 3601 gtagataaaa aagtatttga agaaaatgat attcatgttc acgttcctga aggagctgtt 3661 ccaaaagatg gaccaagtgc tggtatcaca attacaactg ctttaatttc tgcactttca 3721 gataaaccag tttcaaaaga aattggaatg actggtgaaa tcacactaag aggaaatgta 3781 ttgccaattg gtggattaag agaaaaatca atttctgctt caagaagtgg attgaaaaca 3841 atcattattc caaagaaaaa tgaaagagac ttagacgaaa ttccagatga agtaaaagca 3901 aaactaaaaa ttatacctgc tgaaaaatat gaagaggtat ttgcaatagt ttttaaaaca 3961 aaagctgcaa acgacgaaaa ctacgcttta gtagcttaat aaggatccaa actcgagtaa 4021 ggatctccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta 4081 tctgttgttt gtcggtgaac gctctctact agagtcacac tggctcacct tcgggtgggc 4141 ctttctgcgt ttatacctag ggcgttcggc tgcggcgagc ggtatcagct cactcaaagg 4201 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 4261 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 4321 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 4381 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 4441 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 4501 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 4561 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 4621 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 4681 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 4741 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 4801 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 4861 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4921 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg actagtgctt 4981 ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg 5041 agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctcga tatcaaatta 5101 cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg 5161 gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc 5221 ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac 5281 gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc 5341 aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata 5401 tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc 5461 agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc 5521 gtctttcatt gccatacgaa attccggatg agcattcatc aggcgggcaa gaatgtgaat 5581 aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc 5641 cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc 5701 tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt 5761 agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta gtgatcttat 5821 ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtctcatt ttcgccagat 5881 atc //