LOCUS 40924_12645 6090 bp DNA circular SYN 25-JUL-2018 DEFINITION Vector for cold shock-induced expression in E. coli of a protein fused to the Trigger Factor chaperone. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6090) AUTHORS TaKaRa TITLE Direct Submission REFERENCE 2 (bases 1 to 6090) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..6090 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 15..81 /label=cspA promoter /note="promoter of the E. coli cold shock protein cspA gene (Mitta et al., 1997)" protein_bind 84..100 /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)." 5'UTR 122..255 /label=cspA 5'UTR /note="5'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)" CDS 256..270 /label=TEE /note="translation enhancing element for E. coli (Qing et al., 2004)" CDS 271..288 /label=6xHis /note="6xHis affinity tag" CDS 289..1584 /label=Trigger Factor /note="ribosome-associated chaperone from E. coli (Hoffmann et al., 2010)" CDS 1594..1617 /label=HRV 3C site /note="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" CDS 1627..1644 /label=thrombin site /note="thrombin recognition and cleavage site" CDS 1651..1662 /label=Factor Xa site /note="Factor Xa recognition and cleavage site" CDS 1666..1998 /gene="mazF" /label=mazF /note="Endoribonuclease toxin MazF from Escherichia coli (strain K12). Accession#: P0AE70" 3'UTR 2051..2195 /label=cspA 3'UTR /note="3'UTR of the E. coli cold shock protein cspA gene (Mitta et al., 1997)" rep_origin complement(2394..2849) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 2949..3053 /label=AmpR promoter CDS 3054..3911 /label=AmpR /note="beta-lactamase" rep_origin 4085..4673 /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" protein_bind complement(4811..4832) /label=CAP binding site /note="CAP binding activates transcription in the presence of cAMP." CDS complement(4848..5927) /label=lacI /note="lac repressor" promoter complement(5928..6005) /label=lacI promoter ORIGIN 1 aaggaatggt gtggccgatt aatcataaat atgaaaaata attgttgcat cacccgccaa 61 tgcgtggctt aatgcacatc aaattgtgag cggataacaa tttgatgtgc tagcgcatat 121 ccagtgtagt aaggcaagtc ccttcaagag ttatcgttga tacccctcgt agtgcacatt 181 cctttaacgc ttcaaaatct gtaaagcacg ccatatcgcc gaaaggcaca cttaattatt 241 aagaggtaat acaccatgaa tcacaaagtg catcatcatc atcatcacat gcaagtttca 301 gttgaaacca ctcaaggcct tggccgccgt gtaacgatta ctatcgctgc tgacagcatc 361 gagaccgctg ttaaaagcga gctggtcaac gttgcgaaaa aagtacgtat tgacggcttc 421 cgcaagggca aagtgccaat gaatatcgtt gctcagcgtt atggcgcgtc tgtacgccag 481 gacgttctgg gtgacctgat gagccgtaac ttcattgacg ccatcattaa agaaaaaatc 541 aatccggctg gcgcaccgac ttatgttccg ggcgaataca agctgggtga agacttcact 601 tactctgtag agtttgaagt ttatccggaa gttgaactgc aaggtctgga agcgatcgaa 661 gttgaaaaac cgatcgttga agtgaccgac gctgacgttg acggcatgct ggatactctg 721 cgtaaacagc aggcgacctg gaaagaaaaa gacggcgctg ttgaagcaga agaccgcgtg 781 accatcgact tcaccggttc tgtagacggc gaagagttcg aaggcggtaa agcgtctgat 841 ttcgtactgg cgatgggcca gggtcgtatg atcccgggct ttgaagacgg tatcaaaggc 901 cacaaagctg gcgaagagtt caccatcgac gtgaccttcc cggaagaata ccacgcagaa 961 aacctgaaag gtaaagcagc gaaattcgct atcaacctga agaaagttga agagcgtgaa 1021 ctgccggaac tgaccgcaga gttcatcaaa cgtttcggcg ttgaagatgg ttccgtagaa 1081 ggtctgcgcg ctgaagtgcg taaaaacatg gagcgcgagc tgaagagcgc catccgtaac 1141 cgcgttaagt ctcaggcgat cgaaggtctg gtaaaagcta acgacatcga cgtaccggct 1201 gcgctgatcg acagcgaaat cgacgttctg cgtcgccagg ctgcacagcg tttcggtggc 1261 aacgaaaaac aagctctgga actgccgcgc gaactgttcg aagaacaggc taaacgccgc 1321 gtagttgttg gcctgctgct gggcgaagtt atccgcacca acgagctgaa agctgacgaa 1381 gagcgcgtga aaggcctgat cgaagagatg gcttctgcgt acgaagatcc gaaagaagtt 1441 atcgagttct acagcaaaaa caaagaactg atggacaaca tgcgcaatgt tgctctggaa 1501 gaacaggctg ttgaagctgt actggcgaaa gcgaaagtga ctgaaaaaga aaccactttc 1561 aacgagctga tgaaccagca ggcgtccgcg ggtctggaag ttctgttcca ggggccctcc 1621 gcgggtctgg tgccacgcgg tagtggtggt atcgaaggta ggcatatggt aagccgatac 1681 gtacccgata tgggcgatct gatttgggtt gattttgacc cgacaaaagg tagcgagcaa 1741 gctggacatc gtccagctgt tgtcctgagt cctttcatgt acaacaacaa aacaggtatg 1801 tgtctgtgtg ttccttgtac aacgcaatca gctggatatc cgttcgaagt tgttttatcc 1861 ggtcaggaac gtgatggcgt agcgttagct gatcaggtaa aaagtatcgc ctggcgggca 1921 agaggagcaa cgaagaaagg aacagttgcc ccagaggaat tacaactcat taaagccaaa 1981 attaacgtac tgattgggta gctcgaggga tccgaattca agcttgtcga cctgcagtct 2041 agataggtaa tctctgctta aaagcacaga atctaagatc cctgccattt ggcggggatt 2101 tttttatttg ttttcaggaa ataaataatc gatcgcgtaa taaaatctat tattattttt 2161 gtgaagaata aatttgggtg caatgagaat gcgcaggccc tttcgtctcg cgcgtttcgg 2221 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 2281 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 2341 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc ataaaattgt 2401 aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 2461 ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagcccg agatagggtt 2521 gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa 2581 agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac ccaaatcaag 2641 ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt 2701 tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg 2761 agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc 2821 cgcgcttaat gcgccgctac agggcgcgta ctatggttgc tttgacgtat gcggtgtgaa 2881 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gtcaggtggc acttttcggg 2941 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 3001 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 3061 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 3121 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 3181 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 3241 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 3301 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 3361 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 3421 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 3481 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 3541 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 3601 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 3661 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 3721 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 3781 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 3841 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 3901 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 3961 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 4021 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 4081 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 4141 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 4201 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc 4261 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 4321 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 4381 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 4441 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 4501 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 4561 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 4621 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 4681 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atagtcatgc 4741 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc 4801 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc 4861 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 4921 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga 4981 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc 5041 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg 5101 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg 5161 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg 5221 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc 5281 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc 5341 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat 5401 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg 5461 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc 5521 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc 5581 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac 5641 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac 5701 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc 5761 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt 5821 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa 5881 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg 5941 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg 6001 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag 6061 taggttgagg ccgttgagca ccgccgccgc //