LOCUS       sequence-343419-        8334 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a hyperactive PARP1 mutant (A774L 
            destabilizes the autoinhibitory HD subdomain).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8334)
  AUTHORS   Ooi SK, Sato S, Tomomori-Sato C, Zhang Y, Wen Z, Banks CAS, Washburn
            MP, Unruh JR, Florens L, Conaway RC, Conaway JW
  TITLE     Multiple roles for PARP1 in ALC1-dependent nucleosome remodeling.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Sep 7;118(36). pii: 2107277118. doi: 
            10.1073/pnas.2107277118.
  PUBMED    34465625
REFERENCE   2  (bases 1 to 8334)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2021-09-7"; volume: "118(36). pii: 
            2107277118. doi"; pages: " 10.1073/pnas.2107277118"
COMMENT     Sequence Label: pET28_6xHis-PARP1-A774L
FEATURES             Location/Qualifiers
     source          1..8334
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     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 gaggtgctct 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 ctttgctggg
     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
//
