LOCUS       sequence-345352-        6882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a fusion of yeast GAL4 DNA binding domain 
            and human PARP1 CAT domain containing L713F gain-of-function 
            mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6882)
  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 6882)
  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-GAL4-PARP1-CAT-L713F
FEATURES             Location/Qualifiers
     source          1..6882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     884..903
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(923..1000)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1206..1225
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1309..1328
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1309..1327
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1328..1352
                     /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             1367..1389
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1408..1425
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1435..1452
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1456..1488
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1501..1941
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             3055..3072
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3125..3143)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3139..3186
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3223..3678
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3310..3329)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3520..3541
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3770..4585)
                     /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(3770..4585)
                     /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     4493..4512
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4577..4596
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4707..5295
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5196..5215
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5449..5466
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5481..5620
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5546..5565)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5706..5728
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5722..5913)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6722..6882,1..922))
                     /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"
ORIGIN
        1 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
       61 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
      121 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
      181 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
      241 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
      301 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
      361 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
      421 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
      481 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
      541 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
      601 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
      661 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
      721 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
      781 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
      841 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
      901 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
      961 atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc
     1021 ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc
     1081 gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct
     1141 gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc
     1201 ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg
     1261 gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca
     1321 ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta
     1381 agaaggagat ataccatggg cagcagccat catcatcatc atcacagcag cggcctggtg
     1441 ccgcgcggca gccatatggc tagcatgact ggtggacagc aaatgggtcg cggatctacc
     1501 atgaagctac tgtcttctat cgaacaagca tgcgatattt gccgacttaa aaagctcaag
     1561 tgctccaaag aaaaaccgaa gtgcgccaag tgtctgaaga acaactggga gtgtcgctac
     1621 tctcccaaaa ccaaaaggtc tccgctgact agggcacatc tgacagaagt ggaatcaagg
     1681 ctagaaagac tggaacagct atttctactg atttttcctc gagaagacct tgacatgatt
     1741 ttgaaaatgg attctttaca ggatataaaa gcattgttaa caggattatt tgtacaagat
     1801 aatgtgaata aagatgccgt cacagataga ttggcttcag tggagactga tatgcctcta
     1861 acattgagac agcatagaat aagtgcgaca tcatcatcgg aagagagtag taacaaaggt
     1921 caaagacagt tgactgtatc ggctagcgtc gagaccaagt ccaagctccc caagccagtt
     1981 caggacctca tcaagatgat ctttgatgtg gaaagtatga agaaagccat ggtggagtat
     2041 gagatcgacc ttcagaagat gcccttgggg aagctgagca aaaggcagat ccaggccgca
     2101 tactccatct tcagtgaggt ccagcaggcg gtgtctcagg gcagcagcga ctctcagatc
     2161 ctggatctct caaatcgctt ttacaccctg atcccccacg actttgggat gaagaagcct
     2221 ccgctcctga acaatgcaga cagtgtgcag gccaaggcgg aaatgcttga caacctgctg
     2281 gacatcgagg tggcctacag tctgctcagg ggagggtctg atgatagcag caaggatccc
     2341 atcgatgtca actatgagaa gctcaaaact gacattaagg tggttgacag agattctgaa
     2401 gaagccgaga tcatcaggaa gtatgttaag aacactcatg caaccacaca caatgcgtat
     2461 gacttggaag tcatcgatat ctttaagata gagcgtgaag gcgaatgcca gcgttacaag
     2521 ccctttaagc agcttcataa ccgaagattg ctgtggcacg ggtccaggac caccaacttt
     2581 gctgggatcc tgtcccaggg tcttcggata gccccgcctg aagcgcccgt gacaggctac
     2641 atgtttggta aagggatcta tttcgctgac atggtctcca agagtgccaa ctactgccat
     2701 acgtctcagg gagacccaat aggcttaatc ctgttgggag aagttgccct tggaaacatg
     2761 tatgaactga agcacgcttc acatatcagc aagttaccca agggcaagca cagtgtcaaa
     2821 ggtttgggca aaactacccc tgatccttca gctaacatta gtctggatgg tgtagacgtt
     2881 cctcttggga ccgggatttc atctggtgtg aatgacacct ctctactata taacgagtac
     2941 attgtctatg atattgctca ggtaaatctg aagtatctgc tgaaactgaa attcaatttt
     3001 aagacctccc tgtggtaaag atccgcctgc agcaagcttg cggccgcact cgagcaccac
     3061 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     3121 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     3181 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     3241 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     3301 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     3361 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     3421 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     3481 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     3541 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     3601 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     3661 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3721 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     3781 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     3841 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     3901 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     3961 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     4021 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     4081 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     4141 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     4201 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     4261 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     4321 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     4381 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     4441 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     4501 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     4561 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     4621 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4681 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4741 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4801 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4861 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4921 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4981 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5041 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5101 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5161 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     5221 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     5281 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     5341 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     5401 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     5461 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5521 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     5581 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     5641 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     5701 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     5761 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     5821 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     5881 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     5941 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     6001 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     6061 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     6121 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     6181 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     6241 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     6301 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     6361 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     6421 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     6481 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     6541 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     6601 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     6661 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     6721 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     6781 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     6841 agacgggcaa cagctgattg cccttcaccg cctggccctg ag
//
