LOCUS       sequence-347406-        7140 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 7140)
  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 7140)
  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: ARH2-c003
FEATURES             Location/Qualifiers
     source          1..7140
                     /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             1917..1934
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1987..2005)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2001..2048
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2085..2540
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2172..2191)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2382..2403
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2632..3447)
                     /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(2632..3447)
                     /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     3355..3374
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3439..3458
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3569..4157
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4058..4077
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4311..4328
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4343..4482
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4408..4427)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4568..4590
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4584..4775)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5584..6666)
                     /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     6628..6647
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6667..6744)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6950..6969
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7053..7072
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7053..7071
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7072..7096
                     /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             7111..7133
                     /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 atggagaaat tcaaagctgc aatgcttttg ggcagtgtcg gcgacgcatt gggctatcgt
      841 aatgtgtgca aagaaaactc gacagtaggc atgaaaatcc aggaagaatt gcagcgctcg
      901 gggggtctgg atcatttggt gttatccccg ggtgaatggc cagtctctga taacacaatc
      961 atgcacatcg caacagctga agccctgaca acagactact ggtgtctgga tgatctttat
     1021 cgcgagatgg tacgttgtta cgtagaaatt gtagagaaat tgccagaacg tcgtcctgac
     1081 ccagcaacga ttgaagggtg cgcacaattg aagcccaaca attatctttt ggcttggcat
     1141 accccgttca atgaaaaagg ctcaggattt ggtgcagcga cgaaggctat gtgtatcggg
     1201 ttgcgttact ggaagcccga acgtttagag actcttatcg aagtttcagt cgagtgcggg
     1261 cgtatgaccc acaaccatcc cacaggattt ttagggagcc tttgtacggc gctgttcgtg
     1321 agctttgcgg ctcaaggtaa gccccttgtt cagtggggac gtgatatgct tcgcgcagtc
     1381 cccttagcgg aggaatactg tcgcaagacc attcgtcaca cagctgaata tcaggagcac
     1441 tggttttatt ttgaagcaaa atggcagttt tatcttgagg aacgcaagat ttcaaaggat
     1501 agtgagaaca aggcgatctt ccccgataac tacgacgcag aggaacgtga aaagacttac
     1561 cgtaagtggt cgtctgaagg ccgtggaggc cgtcgcggac acgatgctcc catgattgca
     1621 tacgatgcgt tacttgcagc cgggaactca tggacggagc tttgtcaccg tgcgatgttc
     1681 cacggtggcg agagcgcagc gactggtacg attgctggat gtctgtttgg tctgttatac
     1741 ggcctggatt tagtccctaa aggactgtat caagacttag aggataaaga aaaattagaa
     1801 gatttgggtg cagccttgta ccgcctttca actgaggaga aataggacgg tctccagtaa
     1861 aggtggatac ggatccgaat tcgagctccg tcgacaagct tgcggccgca ctcgagcacc
     1921 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1981 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     2041 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     2101 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     2161 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     2221 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     2281 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     2341 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     2401 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     2461 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     2521 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2581 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     2641 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     2701 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     2761 aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt
     2821 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     2881 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2941 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     3001 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     3061 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     3121 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     3181 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     3241 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     3301 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     3361 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     3421 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     3481 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3541 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3601 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3661 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     3721 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3781 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3841 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3901 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3961 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4021 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4081 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4141 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4201 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4261 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4321 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4381 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4441 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     4501 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4561 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     4621 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     4681 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     4741 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     4801 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     4861 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     4921 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     4981 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     5041 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     5101 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     5161 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     5221 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     5281 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     5341 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     5401 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     5461 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     5521 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     5581 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5641 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     5701 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     5761 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     5821 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     5881 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5941 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     6001 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     6061 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     6121 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     6181 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     6241 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     6301 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     6361 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     6421 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     6481 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     6541 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     6601 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     6661 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     6721 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     6781 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     6841 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     6901 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     6961 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     7021 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     7081 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat
//
