LOCUS Exported 8334 bp ds-DNA circular SYN 23-SEP-2021 DEFINITION Bacterial expression of a hyperactive PARP1 mutant (G871L destabilizes the autoinhibitory HD subdomain). ACCESSION . VERSION . KEYWORDS pET28_6xHis-PARP1-G871L SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8334) TITLE PARP and ALC1 plasmids REFERENCE 2 (bases 1 to 8334) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 23, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8334 /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 100..117 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 127..144 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 3199..3216 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(3269..3287) /label=T7 Term /note="T7 terminator, reverse primer" terminator 3283..3330 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 3367..3822 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(3454..3473) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 3664..3685 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(3914..4729) /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 4637..4656 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 4721..4740 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 4851..5439 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 5340..5359 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 5593..5610 /label=L4440 /note="L4440 vector, forward primer" misc_feature 5625..5764 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(5690..5709) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5850..5872 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(5866..6057) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(6866..7948) /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 7910..7929 /label=LacI-R /note="LacI, reverse primer" promoter complement(7949..8026) /gene="lacI" /label=lacI promoter primer_bind 8232..8251 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" ORIGIN 1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc 121 agcggcctgg tgccgcgcgg cagccatatg gcggagtctt ctgataagct gtatcgagtc 181 gagtacgcca agagcgggcg cgcctcttgc aagaaatgca gcgagagcat ccccaaggac 241 tcgctccgga tggccatcat ggtgcagtcg cccatgtttg atggaaaagt cccacactgg 301 taccacttct cctgcttctg gaaggtgggc cactccatcc ggcaccctga cgttgaggtg 361 gatgggttct ctgagcttcg gtgggatgac cagcagaaag tcaagaagac agcggaagct 421 ggaggagtga caggcaaagg ccaggatgga attggtagca aggcagagaa gactctgggt 481 gactttgcag cagagtatgc caagtccaac agaagtacgt gcaaggggtg tatggagaag 541 atagaaaagg gccaggtgcg cctgtccaag aagatggtgg acccggagaa gccacagcta 601 ggcatgattg accgctggta ccatccaggc tgctttgtca agaacaggga ggagctgggt 661 ttccggcccg agtacagtgc gagtcagctc aagggcttca gcctccttgc tacagaggat 721 aaggaagccc tgaagaagca gctcccagga gtcaagagtg aaggaaagag aaaaggcgat 781 gaggtggatg gagtggatga agtggcgaag aagaaatcta aaaaagaaaa agacaaggat 841 agtaagcttg aaaaagccct aaaggctcag aacgacctga tctggaacat caaggacgag 901 ctaaagaaag tgtgttcaac taatgacctg aaggagctac tcatcttcaa caagcagcaa 961 gtgccttctg gggagtcggc gatcttggac cgagtagctg atggcatggt gttcggtgcc 1021 ctccttccct gcgaggaatg ctcgggtcag ctggtcttca agagcgatgc ctattactgc 1081 actggggacg tcactgcctg gaccaagtgt atggtcaaga cacagacacc caaccggaag 1141 gagtgggtaa ccccaaagga attccgagaa atctcttacc tcaagaaatt gaaggttaaa 1201 aagcaggacc gtatattccc cccagaaacc agcgcctccg tggcggccac gcctccgccc 1261 tccacagcct cggctcctgc tgctgtgaac tcctctgctt cagcagataa gccattatcc 1321 aacatgaaga tcctgactct cgggaagctg tcccggaaca aggatgaagt gaaggccatg 1381 attgagaaac tcggggggaa gttgacgggg acggccaaca aggcttccct gtgcatcagc 1441 accaaaaagg aggtggaaaa gatgaataag aagatggagg aagtaaagga agccaacatc 1501 cgagttgtgt ctgaggactt cctccaggac gtctccgcct ccaccaagag ccttcaggag 1561 ttgttcttag cgcacatctt gtccccttgg ggggcagagg tgaaggcaga gcctgttgaa 1621 gttgtggccc caagagggaa gtcaggggct gcgctctcca aaaaaagcaa gggccaggtc 1681 aaggaggaag gtatcaacaa atctgaaaag agaatgaaat taactcttaa aggaggagca 1741 gctgtggatc ctgattctgg actggaacac tctgcgcatg tcctggagaa aggtgggaag 1801 gtcttcagtg ccacccttgg cctggtggac atcgttaaag gaaccaactc ctactacaag 1861 ctccagcttc tggaggacga caaggaaaac aggtattgga tattcaggtc ctggggccgt 1921 gtgggtacgg tgatcggtag caacaaactg gaacagatgc cgtccaagga ggatgccatt 1981 gagcacttca tgaaattata tgaagaaaaa accgggaacg cttggcactc caaaaatttc 2041 acgaagtatc ccaaaaagtt ctaccccctg gagattgact atggccagga tgaagaggca 2101 gtgaagaagc tgacagtaaa tcctggcacc aagtccaagc tccccaagcc agttcaggac 2161 ctcatcaaga tgatctttga tgtggaaagt atgaagaaag ccatggtgga gtatgagatc 2221 gaccttcaga agatgccctt ggggaagctg agcaaaaggc agatccaggc cgcatactcc 2281 atcctcagtg aggtccagca ggcggtgtct cagggcagca gcgactctca gatcctggat 2341 ctctcaaatc gcttttacac cctgatcccc cacgactttg ggatgaagaa gcctccgctc 2401 ctgaacaatg cagacagtgt gcaggccaag gcggaaatgc ttgacaacct gctggacatc 2461 gaggtggcct acagtctgct caggggaggg tctgatgata gcagcaagga tcccatcgat 2521 gtcaactatg agaagctcaa aactgacatt aaggtggttg acagagattc tgaagaagcc 2581 gagatcatca ggaagtatgt taagaacact catgcaacca cacacaatgc gtatgacttg 2641 gaagtcatcg atatctttaa gatagagcgt gaaggcgaat gccagcgtta caagcccttt 2701 aagcagcttc ataaccgaag attgctgtgg cacgggtcca ggaccaccaa ctttgctctg 2761 atcctgtccc agggtcttcg gatagccccg cctgaagcgc ccgtgacagg ctacatgttt 2821 ggtaaaggga tctatttcgc tgacatggtc tccaagagtg ccaactactg ccatacgtct 2881 cagggagacc caataggctt aatcctgttg ggagaagttg cccttggaaa catgtatgaa 2941 ctgaagcacg cttcacatat cagcaagtta cccaagggca agcacagtgt caaaggtttg 3001 ggcaaaacta cccctgatcc ttcagctaac attagtctgg atggtgtaga cgttcctctt 3061 gggaccggga tttcatctgg tgtgaatgac acctctctac tatataacga gtacattgtc 3121 tatgatattg ctcaggtaaa tctgaagtat ctgctgaaac tgaaattcaa ttttaagacc 3181 tccctgtggt aactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc 3241 cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg 3301 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg 3361 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 3421 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 3481 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 3541 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 3601 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 3661 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 3721 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 3781 aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg 3841 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct 3901 catgaattaa ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat 3961 caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac 4021 cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa 4081 catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac 4141 catgagtgac gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt 4201 gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat 4261 tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac 4321 aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac 4381 ctgaatcagg atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga 4441 gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt 4501 ccgtcagcca gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc 4561 catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac 4621 ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg 4681 aatttaatcg cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg 4741 tattactgtt tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag 4801 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 4861 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 4921 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 4981 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 5041 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 5101 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 5161 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 5221 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 5281 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 5341 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 5401 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 5461 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 5521 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 5581 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt 5641 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct 5701 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc 5761 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 5821 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 5881 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga 5941 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa 6001 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga 6061 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag 6121 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg 6181 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca 6241 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca 6301 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg 6361 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct 6421 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc 6481 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc 6541 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc 6601 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg 6661 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat 6721 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga 6781 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag 6841 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 6901 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca 6961 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc 7021 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt 7081 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta 7141 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg 7201 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca 7261 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa 7321 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac 7381 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc 7441 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga 7501 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt 7561 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg 7621 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca 7681 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac 7741 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt 7801 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa 7861 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg 7921 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc 7981 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc 8041 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc 8101 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg 8161 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga 8221 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg 8281 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaat //