LOCUS Exported 5856 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Bacterial expression for structure determination; may not be full ORF. ACCESSION . VERSION . KEYWORDS KEAP1 (KEAP1A-c029) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5856) TITLE SGC Oxford April 2018 plasmids REFERENCE 2 (bases 1 to 5856) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5856 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 1..20 /label=T7 /note="T7 promoter, forward primer" promoter 1..19 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 20..44 /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)." RBS 59..81 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 92..109 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 134..154 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 721..738 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(791..809) /label=T7 Term /note="T7 terminator, reverse primer" terminator 805..852 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 889..1344 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(976..995) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 1186..1207 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(1436..2251) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 2159..2178 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 2243..2262 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 2373..2961 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2862..2881 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3115..3132 /label=L4440 /note="L4440 vector, forward primer" misc_feature 3147..3286 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(3212..3231) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 3372..3394 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(3388..3579) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(4388..5470) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR ALADSLMQLARQVSRLESGQ" primer_bind 5432..5451 /label=LacI-R /note="LacI, reverse primer" promoter complement(5471..5548) /gene="lacI" /label=lacI promoter primer_bind 5754..5773 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgt 121 agatctgggt accgagaacc tgtacttcca atccatgggc aaccgcacct tcagctacac 181 cctggaggat cataccaagc aggcctttgg catcatgaac gagctgcggc tcagccagca 241 gctgtgtgac gtcacactgc aggtcaagta ccaggatgca ccggccgccc agttcatggc 301 ccacaaggtg gtgctggcct catccagccc tgtcttcaag gccatgttca ccaacgggct 361 gcgggagcag ggcatggagg tggtgtccat tgagggtatc caccccaagg tcatggagcg 421 cctcattgaa ttcgcctaca cggcctccat ctccatgggc gagaagtgtg tcctccacgt 481 catgaacggt gctgtcatgt accagatcga cagcgttgtc cgtgcctgca gtgacttcct 541 ggtgcagcag ctggacccca gcaatgccat cggcatcgcc aacttcgctg agcagattgg 601 ctgtgtggag ttgcaccagc gtgcccggga gtacatctac atgcattttg gggagtgaca 661 gtaaaggtgg atacggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag 721 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg 781 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg 841 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta 901 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 961 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 1021 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 1081 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat 1141 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 1201 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc 1261 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 1321 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc 1381 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga 1441 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat 1501 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga 1561 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta 1621 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat 1681 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat 1741 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct 1801 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca 1861 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt 1921 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag 1981 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc 2041 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact 2101 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat 2161 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag 2221 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag 2281 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 2341 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 2401 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 2461 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 2521 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 2581 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 2641 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 2701 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 2761 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 2821 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 2881 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 2941 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 3001 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 3061 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 3121 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc 3181 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa 3241 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc 3301 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc 3361 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc 3421 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg 3481 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa 3541 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg 3601 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt 3661 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg 3721 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga 3781 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt 3841 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca 3901 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat 3961 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga 4021 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc 4081 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc 4141 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa 4201 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat 4261 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc 4321 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc 4381 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 4441 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca 4501 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca 4561 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg 4621 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa 4681 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa 4741 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg 4801 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat 4861 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca 4921 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt 4981 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg 5041 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa 5101 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga 5161 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt 5221 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa 5281 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct 5341 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca 5401 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta 5461 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc 5521 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc 5581 tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat 5641 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc 5701 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg 5761 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt 5821 ccggcgtaga ggatcgagat ctcgatcccg cgaaat //