LOCUS       sequence-349880-        6652 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 6652)
  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 6652)
  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-c010
FEATURES             Location/Qualifiers
     source          1..6652
                     /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             1429..1446
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1499..1517)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1513..1560
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1597..2052
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1684..1703)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1894..1915
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2144..2959)
                     /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(2144..2959)
                     /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     2867..2886
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2951..2970
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3081..3669
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3570..3589
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3823..3840
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3855..3994
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3920..3939)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4080..4102
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4096..4287)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5096..6178)
                     /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     6140..6159
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6179..6256)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6462..6481
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6565..6584
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6565..6583
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6584..6608
                     /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             6623..6645
                     /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 atgttttatg ggactgtttc tagccctgat tcaggtgtgt atgaaatgaa gattggctcc
      841 atcatcttcc aggtggcttc tggagatatc acgaaagaag aggcagatgt gattgtaaat
      901 tcaacatcaa actcattcaa tctcaaagca ggggtctcca aagcaatttt agaatgtgct
      961 ggacaaaatg tagaaaggga atgttctcag caagctcagc agcgcaaaaa tgattatata
     1021 atcaccggag gtggattttt gaggtgcaag aatatcattc atgtaattgg tggaaatgac
     1081 gtcaagagtt cagtttcctc tgttttgcag gagtgtgaaa aaaaaaatta ctcatccatt
     1141 tgcctcccag ccattgggac aggaaatgcc aaacaacacc cagataaggt tgctgaagcc
     1201 ataattgatg ccattgaaga ctttgtccag aaaggatcag cccagtctgt gaaaaaagtt
     1261 aaagttgtta tctttctgcc tcaagtactg gatgtgtttt atgccaacat gaagaaaaga
     1321 gaagggtgac agtaaaggtg gatacggatc cgaataagac ggtctccagt aaaggtggat
     1381 acggatccga attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac
     1441 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1501 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1561 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1621 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1681 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1741 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1801 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1861 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1921 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1981 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     2041 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     2101 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     2161 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     2221 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     2281 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     2341 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     2401 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     2461 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     2521 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     2581 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     2641 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     2701 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     2761 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     2821 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     2881 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     2941 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     3001 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3061 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3121 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3181 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     3241 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3301 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3361 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3421 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3481 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3541 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3601 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3661 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3721 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3781 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3841 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3901 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3961 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     4021 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4081 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     4141 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     4201 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     4261 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     4321 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     4381 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     4441 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4501 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4561 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4621 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4681 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4741 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     4801 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     4861 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     4921 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     4981 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     5041 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     5101 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5161 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     5221 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     5281 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     5341 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     5401 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     5461 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     5521 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     5581 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     5641 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     5701 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     5761 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     5821 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     5881 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     5941 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     6001 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     6061 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     6121 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     6181 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     6241 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     6301 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
     6361 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
     6421 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
     6481 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
     6541 atcgagatct cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata
     6601 acaattcccc tctagaaata attttgttta actttaagaa ggagatatac at
//
