LOCUS pET100/D-TOPO 5764 bp ds-DNA circular SYN 15-AUG-2016DEFINITION synthetic circular DNA ORGANISM synthetic DNA constructREFERENCE 1 (bases 1 to 5764) AUTHORS Biofeng Lab. TITLE Direct SubmissionFEATURES Location/Qualifiers source 1..5764 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 209..227 /note="T7 promoter" /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 228..252 /bound_moiety="lac repressor encoded by lacI" /note="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)." RBS 282..288 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" misc_feature 297..299 /note="ATG" /note="Initiation ATG:" CDS 309..326 /codon_start=1 /product="6xHis affinity tag" /note="6xHis" /translation="HHHHHH" CDS 330..362 /codon_start=1 /product="leader peptide from bacteriophage T7 gene 10 T7 tag (gene 10 leader)" /note="T7 tag" /note="promotes efficient translation in E. coli" /translation="MASMTGGQQMG" CDS 366..389 /codon_start=1 /product="Xpress(TM) epitope tag, including an enterokinase recognition and cleavage site" /note="Xpress(TM) tag" /translation="DLYDDDDK" misc_feature 396..400 /note="TOPO site1" /note="TOPO recognition site1" misc_feature 405..409 /note="TOPO site 2" /note="TOPO recognition site 2" terminator 427..555 /note="T7 terminator" /note="transcription terminator for bacteriophage T7 RNA polymerase" promoter complement(705..733) /note="tet promoter" /note="E. coli promoter for tetracycline efflux protein gene" promoter 856..954 /gene="bla" /note="bla promoter" CDS 955..1815 /codon_start=1 /gene="bla" /product="beta-lactamase" /note="AmpR" /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 2022..2757 /direction=RIGHT /note="pBR322" /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" misc_feature 2760..2899 /note="bom" /note="basis of mobility region from pBR322" CDS complement(3001..3192) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /note="rop" /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4507..5595) /codon_start=1 /gene="lacI" /product="lac repressor" /note="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)." /translation="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA SPRALADSLMQLARQVSRLESGQ" promoter complement(5599..5664) /gene="lacI" /note="lacI promoter"ORIGIN 1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa 61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat 121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta 181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg 241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc 301 ggggttctca tcatcatcat catcatggta tggctagcat gactggtgga cagcaaatgg 361 gtcgggatct gtacgacgat gacgataagg atcatccctt caccaagggc gagctcaacg 421 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat 481 aactagcata accccttggg gcctctaaac gggtcttgag gagttttttg ctgaaaggag 541 gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc 601 tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca 661 cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat 721 gataagctgt caaacatgag aattaattct tgaagacgaa agggcctcgt gatacgccta 781 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 841 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 901 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 961 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 1021 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 1081 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 1141 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt 1201 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1261 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1321 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1381 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 1441 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca 1501 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 1561 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 1621 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 1681 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 1741 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 1801 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 1861 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 1921 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 1981 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 2041 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 2101 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 2161 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 2221 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2281 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2341 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2401 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 2461 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2521 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2581 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 2641 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 2701 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 2761 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc 2821 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg 2881 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc 2941 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg 3001 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc 3061 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt 3121 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc 3181 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc 3241 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact 3301 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac 3361 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca 3421 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag 3481 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt 3541 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag 3601 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg 3661 ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat 3721 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc 3781 tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag 3841 gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg 3901 cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg 3961 atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt 4021 ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg 4081 ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc 4141 agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg 4201 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat 4261 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg 4321 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt 4381 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc 4441 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg 4501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 4561 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag 4621 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg 4681 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat 4741 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg 4801 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag 4861 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat 4921 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt 4981 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc 5041 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg 5101 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac 5161 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat 5221 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc 5281 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat 5341 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag 5401 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc 5461 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac 5521 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg 5581 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa 5641 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca 5701 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg 5761 catg//