LOCUS Exported 7911 bp ds-DNA circular SYN 15-SEP-2021 DEFINITION Bacterial expression of PARP1 lacking WGR domain but containing L713F gain-of-function mutation. ACCESSION . VERSION . KEYWORDS pET28_6xHis-PARP1delWGR-L713F SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7911) TITLE HOLD-Conaway Lab Plasmids REFERENCE 2 (bases 1 to 7911) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 15, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7911 /organism="synthetic DNA construct" /mol_type="other DNA" CDS complement(106..1188) /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 1150..1169 /label=LacI-R /note="LacI, reverse primer" promoter complement(1189..1266) /gene="lacI" /label=lacI promoter primer_bind 1472..1491 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1575..1594 /label=T7 /note="T7 promoter, forward primer" promoter 1575..1593 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1594..1618 /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 1633..1655 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1674..1691 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1701..1718 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" primer_bind complement(4420..4438) /label=T7 Term /note="T7 terminator, reverse primer" terminator 4434..4481 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 4518..4973 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4605..4624) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4815..4836 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(5065..5880) /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 5788..5807 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 5872..5891 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 6002..6590 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6491..6510 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 6744..6761 /label=L4440 /note="L4440 vector, forward primer" misc_feature 6776..6915 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(6841..6860) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 7001..7023 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(7017..7208) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" ORIGIN 1 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat 61 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc 121 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 181 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg 241 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc 301 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc 361 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat 421 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat 481 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc 541 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg 601 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa 661 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt 721 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc 781 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg 841 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga 901 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga 961 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc 1021 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt 1081 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata 1141 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct 1201 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat 1261 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta 1321 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc 1381 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga 1441 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa 1501 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct 1561 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc 1621 tctagaaata attttgttta actttaagaa ggagatatac catgggcagc agccatcatc 1681 atcatcatca cagcagcggc ctggtgccgc gcggcagcca tatgatggcg gagtcttctg 1741 ataagctgta tcgagtcgag tacgccaaga gcgggcgcgc ctcttgcaag aaatgcagcg 1801 agagcatccc caaggactcg ctccggatgg ccatcatggt gcagtcgccc atgtttgatg 1861 gaaaagtccc acactggtac cacttctcct gcttctggaa ggtgggccac tccatccggc 1921 accctgacgt tgaggtggat gggttctctg agcttcggtg ggatgaccag cagaaagtca 1981 agaagacagc ggaagctgga ggagtgacag gcaaaggcca ggatggaatt ggtagcaagg 2041 cagagaagac tctgggtgac tttgcagcag agtatgccaa gtccaacaga agtacgtgca 2101 aggggtgtat ggagaagata gaaaagggcc aggtgcgcct gtccaagaag atggtggacc 2161 cggagaagcc acagctaggc atgattgacc gctggtacca tccaggctgc tttgtcaaga 2221 acagggagga gctgggtttc cggcccgagt acagtgcgag tcagctcaag ggcttcagcc 2281 tccttgctac agaggataag gaagccctga agaagcagct cccaggagtc aagagtgaag 2341 gaaagagaaa aggcgatgag gtggatggag tggatgaagt ggcgaagaag aaatctaaaa 2401 aagaaaaaga caaggatagt aagcttgaaa aagccctaaa ggctcagaac gacctgatct 2461 ggaacatcaa ggacgagcta aagaaagtgt gttcaactaa tgacctgaag gagctactca 2521 tcttcaacaa gcagcaagtg ccttctgggg agtcggcgat cttggaccga gtagctgatg 2581 gcatggtgtt cggtgccctc cttccctgcg aggaatgctc gggtcagctg gtcttcaaga 2641 gcgatgccta ttactgcact ggggacgtca ctgcctggac caagtgtatg gtcaagacac 2701 agacacccaa ccggaaggag tgggtaaccc caaaggaatt ccgagaaatc tcttacctca 2761 agaaattgaa ggttaaaaag caggaccgta tattcccccc agaaaccagc gcctccgtgg 2821 cggccacgcc tccgccctcc acagcctcgg ctcctgctgc tgtgaactcc tctgcttcag 2881 cagataagcc attatccaac atgaagatcc tgactctcgg gaagctgtcc cggaacaagg 2941 atgaagtgaa ggccatgatt gagaaactcg gggggaagtt gacggggacg gccaacaagg 3001 cttccctgtg catcagcacc aaaaaggagg tggaaaagat gaataagaag atggaggaag 3061 taaaggaagc caacatccga gttgtgtctg aggacttcct ccaggacgtc tccgcctcca 3121 ccaagagcct tcaggagttg ttcttagcgc acatcttgtc cccttggggg gcagaggtga 3181 aggcagagcc tgttgaagtt gtggccccaa gagggaagtc aggggctgcg ctctccaaaa 3241 aaagcaaggg ccaggtcaag gaggaaggta tcaacaaatc tgaaaccaag tccaagctcc 3301 ccaagccagt tcaggacctc atcaagatga tctttgatgt ggaaagtatg aagaaagcca 3361 tggtggagta tgagatcgac cttcagaaga tgcccttggg gaagctgagc aaaaggcaga 3421 tccaggccgc atactccatc ttcagtgagg tccagcaggc ggtgtctcag ggcagcagcg 3481 actctcagat cctggatctc tcaaatcgct tttacaccct gatcccccac gactttggga 3541 tgaagaagcc tccgctcctg aacaatgcag acagtgtgca ggccaaggcg gaaatgcttg 3601 acaacctgct ggacatcgag gtggcctaca gtctgctcag gggagggtct gatgatagca 3661 gcaaggatcc catcgatgtc aactatgaga agctcaaaac tgacattaag gtggttgaca 3721 gagattctga agaagccgag atcatcagga agtatgttaa gaacactcat gcaaccacac 3781 acaatgcgta tgacttggaa gtcatcgata tctttaagat agagcgtgaa ggcgaatgcc 3841 agcgttacaa gccctttaag cagcttcata accgaagatt gctgtggcac gggtccagga 3901 ccaccaactt tgctgggatc ctgtcccagg gtcttcggat agccccgcct gaagcgcccg 3961 tgacaggcta catgtttggt aaagggatct atttcgctga catggtctcc aagagtgcca 4021 actactgcca tacgtctcag ggagacccaa taggcttaat cctgttggga gaagttgccc 4081 ttggaaacat gtatgaactg aagcacgctt cacatatcag caagttaccc aagggcaagc 4141 acagtgtcaa aggtttgggc aaaactaccc ctgatccttc agctaacatt agtctggatg 4201 gtgtagacgt tcctcttggg accgggattt catctggtgt gaatgacacc tctctactat 4261 ataacgagta cattgtctat gatattgctc aggtaaatct gaagtatctg ctgaaactga 4321 aattcaattt taagacctcc ctgtggtaac tcgaccacca ccaccactga gatccggctg 4381 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat 4441 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat 4501 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 4561 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 4621 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 4681 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 4741 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 4801 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 4861 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 4921 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc 4981 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 5041 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa 5101 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg 5161 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc 5221 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag 5281 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc 5341 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac 5401 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa 5461 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac 5521 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat 5581 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag 5641 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac 5701 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata 5761 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc 5821 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat 5881 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc 5941 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 6001 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 6061 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 6121 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact 6181 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 6241 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 6301 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 6361 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 6421 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 6481 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 6541 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca 6601 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg 6661 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc 6721 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga 6781 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag 6841 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac 6901 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 6961 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 7021 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg 7081 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc 7141 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt 7201 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg 7261 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa 7321 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag 7381 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat 7441 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt 7501 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag 7561 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa 7621 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc 7681 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg 7741 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc 7801 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct 7861 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat g //