LOCUS       sequence-347991-        7668 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 7668)
  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 7668)
  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: PARG-c002
FEATURES             Location/Qualifiers
     source          1..7668
                     /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             2445..2462
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2515..2533)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2529..2576
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2613..3068
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2700..2719)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2910..2931
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3160..3975)
                     /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(3160..3975)
                     /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     3883..3902
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3967..3986
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4097..4685
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4586..4605
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4839..4856
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4871..5010
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4936..4955)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5096..5118
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5112..5303)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6112..7194)
                     /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     7156..7175
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7195..7272)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7478..7497
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7581..7600
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7581..7599
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7600..7624
                     /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             7639..7661
                     /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 atgtctccag ataagaagtg gcttggaact cccattgagg agatgagaag aatgcctcgg
      841 tgtgggatcc ggctgcctct cttgagacca tctgccaatc acacagtaac tattcgggta
      901 gatcttttgc gagcaggaga agttcctaaa ccttttccaa cacattataa agatttgtgg
      961 gataacaagc atgttaaaat gccttgttca gaacaaaatt tgtacccagt ggaagatgag
     1021 aatggtgagc gaactgcggg gagccggtgg gagctcattc agactgcact tctcaacaaa
     1081 tttacacgac cccaaaactt gaaggatgct attctgaaat acaatgtggc atattctaag
     1141 aaatgggact ttacagcttt gatcgatttc tgggataagg tacttgaaga agcagaagct
     1201 caacatttat atcagtccat cttgcctgat atggtgaaaa ttgcactctg tctgccaaat
     1261 atttgcaccc agccaatacc actcctgaaa cagaagatga atcattccat cacaatgtcg
     1321 caggaacaga ttgccagtct tttagctaat gctttcttct gcacatttcc acgacgaaat
     1381 gctaagatga aatcggagta ttctagttac ccagacatta acttcaatcg attgtttgag
     1441 ggacgttcat caaggaaacc ggagaaactt aaaacgctct tctgctactt tagaagagtc
     1501 acagagaaaa aacctactgg gttggtgaca tttacaagac agagtcttga agattttcca
     1561 gaatgggaaa gatgtgaaaa acccttgaca cgattgcatg tcacttacga aggtaccata
     1621 gaagaaaatg gccaaggcat gctacaggtg gattttgcaa atcgttttgt tggaggtggt
     1681 gtaaccagtg caggacttgt gcaagaagaa atccgctttt taatcaatcc tgagttgatt
     1741 atttcacggc tcttcactga ggtgctggat cacaatgaat gtctaattat cacaggtact
     1801 gagcagtaca gtgaatacac aggctatgct gagacatatc gttggtcccg gagccacgaa
     1861 gatgggagtg aaagggacga ctggcagcgg cgctgcactg agatcgttgc catcgatgct
     1921 cttcacttca gacgctacct cgatcagttt gtgcctgaga aaatgagacg cgagctgaac
     1981 aaggcttact gtggatttct ccgtcctgga gtttcttcag agaatctttc tgcagtggcc
     2041 acaggaaact ggggctgtgg tgcctttggg ggtgatgcca ggttaaaagc cttaatacag
     2101 atattggcag ctgctgcagc tgagcgagat gtggtttatt tcacctttgg ggactcagaa
     2161 ttgatgagag acatttacag catgcacatt ttccttactg aaaggaaact cactgttgga
     2221 gatgtgtata agctgttgct acgatactac aatgaagaat gcagaaactg ttccacccct
     2281 ggaccagaca tcaagcttta tccattcata taccatgctg tcgagtcctg tgcagagacc
     2341 gctgaccatt cagggcaaag gacagggacc taagacggtc tccagtaaag gtggatacgg
     2401 atccgaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
     2461 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     2521 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     2581 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     2641 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     2701 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     2761 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2821 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     2881 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     2941 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3001 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     3061 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3121 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     3181 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     3241 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     3301 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     3361 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     3421 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     3481 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     3541 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     3601 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     3661 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     3721 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     3781 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     3841 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     3901 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     3961 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     4021 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4081 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4141 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4201 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     4261 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4321 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4381 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4441 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4501 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4561 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4621 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4681 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4741 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4801 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4861 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4921 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     4981 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     5041 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5101 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     5161 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     5221 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     5281 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     5341 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     5401 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     5461 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     5521 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     5581 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     5641 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     5701 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     5761 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     5821 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     5881 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     5941 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     6001 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     6061 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     6121 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6181 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     6241 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     6301 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     6361 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     6421 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     6481 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     6541 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     6601 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     6661 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     6721 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     6781 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     6841 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     6901 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     6961 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     7021 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     7081 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     7141 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     7201 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     7261 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     7321 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     7381 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     7441 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     7501 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     7561 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     7621 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacat
//
