LOCUS       sequence-348267-        6672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of recombinant fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6672)
  AUTHORS   Sowa ST, Galera-Prat A, Wazir S, Alanen HI, Maksimainen MM, Lehtio L
  TITLE     A molecular toolbox for ADP-ribosyl binding proteins.
  JOURNAL   Cell Rep Methods. 2021 Dec 20;1(8):100121. doi: 
            10.1016/j.crmeth.2021.100121. Epub 2021 Nov 11.
  PUBMED    34786571
REFERENCE   2  (bases 1 to 6672)
  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: "Cell Rep 
            Methods."; date: "2021-12-20"; volume: "1(8):100121. doi"; pages: " 
            10.1016/j.crmeth.2021.100121. Epub 2021 Nov 11"
COMMENT     Sequence Label: PARP9-c024
FEATURES             Location/Qualifiers
     source          1..6672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..21
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(22..24,25..735)
                     /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(64..85)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(325..344)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     672..693
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             760..780
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1449..1466
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1519..1537)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1533..1580
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1617..2072
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1704..1723)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1914..1935
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2164..2979)
                     /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(2164..2979)
                     /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     2887..2906
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2971..2990
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3101..3689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3590..3609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3843..3860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3875..4014
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3940..3959)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4100..4122
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4116..4307)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5116..6198)
                     /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     6160..6179
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6199..6276)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6482..6501
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6585..6604
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6585..6603
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6604..6628
                     /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             6643..6665
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgcaccatc atcatcatca tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
       61 atcctggtcg agctggacgg cgacgtaaac ggccacaggt tcagcgtgtc cggcgagggc
      121 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      181 cccgtgccct ggcccaccct cgtgaccacc ctgacctggg gcgtgcagtg cttcgcccgc
      241 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      301 caggagcgta ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      361 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
      421 ggcaacatcc tggggcacaa gctggagtac aacgccatca gcgacaacgt ctatatcacc
      481 gccgacaagc agaagaacgg catcaaggcc cacttcaaga tccgccacaa catcgaggac
      541 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
      601 ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag
      661 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
      721 gacgagctgt acaagtcttc tggtgtagat ctgggtaccg agaacttgta cttccaatcc
      781 atgggcaaca gcaaatctct gcaagtgttc agaaaaatgc tgactcctag gatagagtta
      841 tcagtctgga aagatgacct caccacacat gctgttgatg ctgtggtgaa tgcagccaat
      901 gaagatcttc tgcatggggg aggcctggcc ctggccctgg taaaagctgg tggatttgaa
      961 atccaagaag agagcaaaca gtttgttgcc agatatggta aagtgtcagc tggtgagata
     1021 gctgtcacgg gagcagggag gcttccctgc aaacagatca tccatgctgt tgggcctcgg
     1081 tggatggaat gggataaaca gggatgtact ggaaagctgc agagggccat tgtaagtatt
     1141 ctgaattatg tcatctataa aaatactcac attaagacag tagcaattcc agccttgagc
     1201 tctgggattt ttcagttccc tctgaatttg tgtacaaaga ctattgtaga gactatccgg
     1261 gttagtttgc aagggaagcc aatgatgagt aatttgaaag aaattcacct ggtgagcaat
     1321 gaggacccta ctgttgctgc ctttaaagct gcttcagaat tcatcctagg gaagtaggac
     1381 ggtctccagt aaaggtggat acggatccga attcgagctc cgtcgacaag cttgcggccg
     1441 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     1501 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1561 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1621 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1681 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1741 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1801 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1861 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1921 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1981 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     2041 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     2101 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     2161 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     2221 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     2281 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     2341 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     2401 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     2461 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     2521 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     2581 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     2641 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     2701 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     2761 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     2821 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     2881 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     2941 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     3001 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3061 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3121 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3181 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3241 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3301 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3361 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3421 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3481 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3541 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3601 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3661 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3721 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3781 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3841 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3901 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     3961 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     4021 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     4081 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     4141 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     4201 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     4261 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     4321 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     4381 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     4441 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     4501 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     4561 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4621 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4681 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4741 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     4801 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     4861 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     4921 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     4981 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     5041 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     5101 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5161 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     5221 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     5281 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     5341 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     5401 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     5461 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5521 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5581 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5641 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     5701 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     5761 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     5821 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     5881 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     5941 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     6001 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     6061 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     6121 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     6181 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     6241 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     6301 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     6361 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     6421 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     6481 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     6541 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     6601 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     6661 ggagatatac at
//
