LOCUS Exported 7361 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Bacterial expression of human Parkin. ACCESSION . VERSION . KEYWORDS Human Parkin (1-465) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7361) AUTHORS Wauer T, Komander D TITLE Structure of the human Parkin ligase domain in an autoinhibited state. JOURNAL EMBO J. 2013 Jul 31;32(15):2099-112. doi: 10.1038/emboj.2013.125. Epub 2013 May 31. PUBMED 23727886 REFERENCE 2 (bases 1 to 7361) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7361 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 61..80 /label=T7 /note="T7 promoter, forward primer" promoter 61..79 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 80..104 /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 119..141 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 154..171 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 172..825 /codon_start=1 /product="glutathione S-transferase from Schistosoma japonicum" /label=GST /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK" primer_bind 783..805 /label=pGEX 5' /note="pGEX vectors, Glutathione-S-transferase, forward primer" CDS 844..867 /codon_start=1 /product="recognition and cleavage site for human rhinovirus 3C and PreScission proteases" /label=HRV 3C site /translation="LEVLFQGP" CDS 2286..2303 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(2356..2374) /label=T7 Term /note="T7 terminator, reverse primer" terminator 2370..2417 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 2454..2909 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2541..2560) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2751..2772 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3001..3816) /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 3724..3743 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 3808..3827 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 3938..4526 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 4427..4446 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 4680..4697 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4712..4851 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4777..4796) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4937..4959 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4953..5144) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5953..7035) /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 6997..7016 /label=LacI-R /note="LacI, reverse primer" promoter complement(7036..7113) /gene="lacI" /label=lacI promoter primer_bind 7319..7338 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat 61 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 121 tgtttaactt taagaaggag atataccatg gcacatcacc atcaccatca catgtcccct 181 atactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct tttggaatat 241 cttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa atggcgaaac 301 aaaaagtttg aattgggttt ggagtttccc aatcttcctt attatattga tggtgatgtt 361 aaattaacac agtctatggc catcatacgt tatatagctg acaagcacaa catgttgggt 421 ggttgtccaa aagagcgtgc agagatttca atgcttgaag gagcggtttt ggatattaga 481 tacggtgttt cgagaattgc atatagtaaa gactttgaaa ctctcaaagt tgattttctt 541 agcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa aacatattta 601 aatggtgatc atgtaaccca tcctgacttc atgttgtatg acgctcttga tgttgtttta 661 tacatggacc caatgtgcct ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt 721 gaagctatcc cacaaattga taagtacttg aaatccagca agtatatagc atggcctttg 781 cagggctggc aagccacgtt tggtggtggc gaccatcctc caaaatcgga tctgagcagc 841 ggtctggaag ttctgtttca gggcccgatg attgtttttg tgcgttttaa ctcgtcccac 901 ggttttccgg ttgaagtcga tagcgatacc agcattttcc aactgaaaga agtcgtcgca 961 aaacgtcagg gcgtgccggc agatcaactg cgtgtgattt ttgccggtaa agaactgcgc 1021 aacgactgga ccgttcagaa ttgcgatctg gaccagcaaa gcattgttca tatcgtccag 1081 cgtccgtggc gcaaaggcca agaaatgaac gcaaccggcg gtgatgaccc gcgtaatgca 1141 gccggcggtt gtgaacgtga accgcagtct ctgacgcgcg ttgatctgag ctctagtgtc 1201 ctgccgggtg acagtgtggg tctggcagtt attctgcaca ccgattcccg taaagactca 1261 ccgccggcag gtagcccggc tggtcgttcg atctacaaca gcttctatgt gtactgcaaa 1321 ggcccgtgtc agcgtgtcca accgggtaaa ctgcgcgtgc agtgctcgac ctgtcgtcaa 1381 gcaacgctga ccctgacgca gggtccgagc tgctgggatg acgttctgat cccgaatcgt 1441 atgtctggcg aatgccagag tccgcattgt ccgggtacga gcgcagaatt tttctttaaa 1501 tgtggtgctc atccgacctc cgataaagaa acgtcagtgg cactgcacct gattgctacc 1561 aacagtcgta atattacgtg catcacctgt acggatgttc gctctccggt gctggttttc 1621 cagtgcaaca gtcgtcatgt catctgcctg gactgttttc acctgtactg tgttacccgt 1681 ctgaatgatc gccagttcgt ccacgacccg caactgggct attccctgcc gtgcgttgcg 1741 ggttgtccga actcactgat taaagaactg catcactttc gtatcctggg cgaagaacag 1801 tataatcgct accagcaata tggtgcagaa gaatgcgtgc tgcagatggg cggtgttctg 1861 tgcccgcgtc cgggttgtgg tgcaggtctg ctgccggaac cggatcagcg taaagtgacc 1921 tgcgaaggcg gtaacggtct gggctgtggt ttcgcgtttt gccgcgaatg taaagaagcc 1981 taccatgaag gcgaatgctc ggcggtcttc gaagccagcg gcaccacgac ccaggcatat 2041 cgtgtggatg aacgcgcagc tgaacaagct cgctgggaag cggcctctaa agaaaccatt 2101 aagaaaacca ccaaaccgtg cccgcgttgt catgtcccgg tggagaaaaa cggcggttgc 2161 atgcacatga aatgtccgca gccgcaatgc cgtctggaat ggtgttggaa ctgcggctgc 2221 gaatggaatc gtgtgtgtat gggtgaccac tggtttgatg tgtaaagctt tctagaccat 2281 ttaaacacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg 2341 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg 2401 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc 2461 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact 2521 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc 2581 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt 2641 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc 2701 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt 2761 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat 2821 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa 2881 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga 2941 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc 3001 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat 3061 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca 3121 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc 3181 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac 3241 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca 3301 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg 3361 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga 3421 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata 3481 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc 3541 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt 3601 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa 3661 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac 3721 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg 3781 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat 3841 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact 3901 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 3961 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 4021 aagagctaac aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 4081 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 4141 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 4201 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 4261 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 4321 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 4381 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 4441 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 4501 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 4561 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 4621 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 4681 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc 4741 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata 4801 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac 4861 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 4921 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 4981 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct 5041 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg 5101 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa 5161 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata 5221 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg 5281 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat 5341 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata 5401 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc 5461 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg 5521 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc 5581 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc 5641 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag 5701 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg 5761 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca 5821 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg 5881 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta 5941 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 6001 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct 6061 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg 6121 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa 6181 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc 6241 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt 6301 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa 6361 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt 6421 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc 6481 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc 6541 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa 6601 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata 6661 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc 6721 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg 6781 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac 6841 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt 6901 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg 6961 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa 7021 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat 7081 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg 7141 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa 7201 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca 7261 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg 7321 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc g //