LOCUS       sequence-434365-        6837 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression vector for E. coli producing PARP1 catalytic 
            protein (without Regulatory domain); with N-terminal CFP fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6837)
  AUTHORS   Dhakar SS, Galera-Prat A, Lehtio L
  TITLE     High-throughput screening assay for PARP-HPF1 interaction inhibitors
            to affect DNA damage repair.
  JOURNAL   Sci Rep. 2024 Feb 16;14(1):3875. doi: 10.1038/s41598-024-54123-8.
  PUBMED    38365924
REFERENCE   2  (bases 1 to 6837)
  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: "Sci Rep."; 
            date: "2024-02-16"; volume: "14(1):3875. doi"; pages: " 
            10.1038/s41598-024-54123-8"
COMMENT     Sequence Label: PARP1-c009
FEATURES             Location/Qualifiers
     source          1..6837
                     /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             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(110..112,113..823)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKA
                     HFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(152..173)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(413..432)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     760..781
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             848..868
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1702..1719
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1772..1790)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1786..1833
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1870..2325
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1957..1976)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2167..2188
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2417..3232)
                     /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(2417..3232)
                     /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     3140..3159
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3224..3243
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3354..3942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3843..3862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4096..4113
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4128..4267
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4193..4212)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4353..4375
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4369..4560)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5369..6451)
                     /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     6413..6432
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6452..6529)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6735..6754
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatg tgagcaaggg
      121 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
      181 ccacaggttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
      241 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
      301 gacctggggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt
      361 caagtccgcc atgcccgaag gctacgtcca ggagcgtacc atcttcttca aggacgacgg
      421 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
      481 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
      541 cgccatcagc gacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccca
      601 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
      661 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
      721 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
      781 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtcttctg gtgtagatct
      841 gggtaccgag aacttgtact tccaatccat gaccaagtcc aagctcccca agccagttca
      901 ggacctcatc aagatgatct ttggatcggg atcggggtca ggcggtgatc ccatcgatgt
      961 caactatgag aagctcaaaa ctgacattaa ggtggttgac agagattctg aagaagccga
     1021 gatcatcagg aagtatgtta agaacactca tgcaaccaca cacaatgcgt atgacttgga
     1081 agtcatcgat atctttaaga tagagcgtga aggcgaatgc cagcgttaca agccctttaa
     1141 gcagcttcat aaccgaagat tgctgtggca cgggtccagg accaccaact ttgctgggat
     1201 cctgtcccag ggtcttcgga tagccccgcc tgaagcgccc gtgacaggct acatgtttgg
     1261 taaagggatc tatttcgctg acatggtctc caagagtgcc aactactgcc atacgtctca
     1321 gggagaccca ataggcttaa tcctgttggg agaagttgcc cttggaaaca tgtatgaact
     1381 gaagcacgct tcacatatca gcaagttacc caagggcaag cacagtgtca aaggtttggg
     1441 caaaactacc cctgatcctt cagctaacat tagtctggat ggtgtagacg ttcctcttgg
     1501 gaccgggatt tcatctggtg tgaatgacac ctctctacta tataacgagt acattgtcta
     1561 tgatattgct caggtaaatc tgaagtatct gctgaaactg aaattcaatt ttaagacctc
     1621 cctgtggtaa gacggtctcc agtaaaggtg gatacggatc cgaattcgag ctccgtcgac
     1681 aagcttgcgg ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa
     1741 gcccgaaagg aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt
     1801 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg
     1861 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1921 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1981 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2041 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2101 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     2161 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     2221 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     2281 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     2341 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2401 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
     2461 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
     2521 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
     2581 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
     2641 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
     2701 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
     2761 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
     2821 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     2881 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     2941 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     3001 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     3061 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     3121 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     3181 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     3241 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     3301 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     3361 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     3421 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     3481 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     3541 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     3601 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     3661 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     3721 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3781 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3841 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3901 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3961 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4021 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4081 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4141 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     4201 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     4261 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     4321 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     4381 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     4441 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     4501 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     4561 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     4621 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     4681 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     4741 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     4801 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     4861 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     4921 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     4981 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     5041 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5101 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5161 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5221 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5281 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5341 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5401 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5461 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5521 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     5581 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     5641 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     5701 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     5761 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     5821 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     5881 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     5941 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6001 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6061 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6121 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6181 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6241 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6301 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6361 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6421 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6481 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     6541 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     6601 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     6661 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     6721 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     6781 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaat
//
