LOCUS       sequence-349917-        6645 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 6645)
  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 6645)
  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: Parp14-c012
FEATURES             Location/Qualifiers
     source          1..6645
                     /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             1422..1439
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1492..1510)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1506..1553
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1590..2045
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1677..1696)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1887..1908
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2137..2952)
                     /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(2137..2952)
                     /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     2860..2879
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2944..2963
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3074..3662
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3563..3582
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3816..3833
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3848..3987
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3913..3932)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4073..4095
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4089..4280)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5089..6171)
                     /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     6133..6152
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6172..6249)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6455..6474
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6558..6577
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6558..6576
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6577..6601
                     /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             6616..6638
                     /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 atgtgggaaa aaggaagcct ggtgtccccg ggaggcctgc agatgctgtt ggtgaaagag
      841 ggtgtgcaga atgctaagac cgatgttgtt gtcaactccg ttcccttgga tctcgtgctt
      901 agtagagggc ctctttctaa gtccctcttg gaaaaagctg gaccagagct ccaggaggaa
      961 ttggacacag ttggacaagg ggtggctgtc agcatgggca cagtgctcaa aaccagcagc
     1021 tggaatctgg actgtcgcta tgtgcttcac gtggtagctc cggagtggag aaatggtagc
     1081 acatcttcac tcaagataat ggaagacata atcagagaat gtatggagat cactgagagc
     1141 ttgtccttaa aatcaattgc atttccagca ataggaacag gaaacttggg atttcctaaa
     1201 aacatattcg ctgaattaat catttcagag gtgttcaaat ttagtagcaa gaatcagctg
     1261 aaaactttac aagaggttca ctttctgctg cacccgagtg atcatgaaaa tattcaggca
     1321 ttttcagatg aatttgccag aagggcttag gacggtctcc agtaaaggtg gatacggatc
     1381 cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact
     1441 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     1501 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     1561 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     1621 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1681 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1741 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     1801 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     1861 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     1921 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     1981 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     2041 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2101 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     2161 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     2221 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     2281 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     2341 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     2401 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     2461 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     2521 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     2581 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     2641 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     2701 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     2761 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     2821 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     2881 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     2941 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     3001 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3061 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3121 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3181 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     3241 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3301 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3361 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3421 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3481 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3541 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3601 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3661 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3721 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3781 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3841 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     3901 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     3961 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4021 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4081 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     4141 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     4201 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     4261 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     4321 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     4381 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     4441 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     4501 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     4561 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     4621 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     4681 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     4741 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     4801 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     4861 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     4921 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     4981 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     5041 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     5101 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5161 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     5221 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     5281 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     5341 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     5401 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     5461 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     5521 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     5581 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     5641 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     5701 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     5761 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     5821 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     5881 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     5941 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     6001 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     6061 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     6121 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     6181 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     6241 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     6301 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     6361 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     6421 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     6481 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     6541 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     6601 ccctctagaa ataattttgt ttaactttaa gaaggagata tacat
//
