LOCUS       sequence-347989-        6633 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 6633)
  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 6633)
  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-c020
FEATURES             Location/Qualifiers
     source          1..6633
                     /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             1410..1427
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1480..1498)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1494..1541
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1578..2033
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1665..1684)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1875..1896
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2125..2940)
                     /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(2125..2940)
                     /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     2848..2867
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2932..2951
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3062..3650
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3551..3570
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3804..3821
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3836..3975
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3901..3920)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4061..4083
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4077..4268)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5077..6159)
                     /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     6121..6140
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6160..6237)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6443..6462
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6546..6565
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6546..6564
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6565..6589
                     /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             6604..6626
                     /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 atgaatgcaa tggtcgtgaa caacctgacc ctccagattg tccagggcca cattgaatgg
      841 cagacggcag atgtaattgt taattctgta aacccacatg atattacagt tggacctgtg
      901 gcaaagtcaa ttctacaaca agcaggagtt gaaatgaaat cggaatttct tgccacaaag
      961 gctaaacagt ttcaacggtc ccagttggta ctggtcacaa aaggatttaa cttgttctgt
     1021 aaatatatat accatgtact gtggcattca gaatttccta aacctcagat attaaaacat
     1081 gcaatgaagg agtgtttgga aaaatgcatt gagcaaaata taacttccat ttcctttcct
     1141 gcccttggga ctggaaacat ggaaataaag aaggaaacag cagcagagat tttgtttgat
     1201 gaagttttaa catttgccaa agaccatgta aaacaccagt taactgtaaa atttgtgatc
     1261 tttccaacag atttggagat atataaggct ttcagttctg aaatggcaaa gaggtccaag
     1321 atgctgagtt tgaactgaga cggtctccag taaaggtgga tacggatccg aattcgagct
     1381 ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
     1441 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1501 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1561 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     1621 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     1681 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     1741 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     1801 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     1861 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     1921 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     1981 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     2041 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2101 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     2161 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     2221 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     2281 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     2341 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     2401 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     2461 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     2521 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     2581 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     2641 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     2701 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     2761 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     2821 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     2881 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     2941 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     3001 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3061 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3121 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3181 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3241 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3301 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3361 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3421 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3481 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3541 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     3601 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     3661 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     3721 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     3781 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     3841 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     3901 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     3961 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4021 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4081 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     4141 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     4201 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     4261 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     4321 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     4381 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4441 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     4501 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     4561 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     4621 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     4681 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     4741 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     4801 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     4861 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     4921 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     4981 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     5041 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5101 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5161 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     5221 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     5281 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     5341 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     5401 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     5461 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     5521 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     5581 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     5641 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     5701 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     5761 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     5821 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     5881 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     5941 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     6001 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     6061 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     6121 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     6181 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     6241 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6301 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     6361 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     6421 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     6481 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     6541 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     6601 aattttgttt aactttaaga aggagatata cat
//
