LOCUS       sequence-347408-        7149 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 7149)
  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 7149)
  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: ARH1-c003
FEATURES             Location/Qualifiers
     source          1..7149
                     /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             1926..1943
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1996..2014)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2010..2057
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2094..2549
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2181..2200)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2391..2412
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2641..3456)
                     /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(2641..3456)
                     /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     3364..3383
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3448..3467
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3578..4166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4067..4086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4320..4337
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4352..4491
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4417..4436)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4577..4599
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4593..4784)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5593..6675)
                     /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     6637..6656
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6676..6753)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6959..6978
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7062..7081
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7062..7080
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7081..7105
                     /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             7120..7142
                     /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 atggagaagt atgtagccgc gatggtgctt tctgccgctg gtgacgccct tggatattat
      841 aacggaaagt gggagttctt acaggatggt gaaaagatcc atcgccaatt agcacaactt
      901 ggcgggcttg atgcattgga cgttggacgt tggcgcgtgt ctgatgatac ggtaatgcat
      961 cttgctacgg cagaagcgct tgtagaagca gggaaagcgc ctaagttaac tcagttatat
     1021 tacctgttgg caaagcacta tcaagactgt atggaggata tggacggtcg tgcccccgga
     1081 ggagcgagcg tacacaacgc catgcaactg aagcccggaa aacctaatgg ctggcgtatt
     1141 ccattcaact cacatgaagg gggatgcgga gctgccatgc gtgcaatgtg catcggcttg
     1201 cgtttcccgc atcattccca acttgatacg ctgatccaag tttccatcga gtctgggcgc
     1261 atgactcatc accatcctac cgggtacttg ggagccttgg cgtctgcgct ttttaccgcg
     1321 tatgcggtta acagtcgtcc acctcttcaa tggggtaagg ggcttatgga actgcttccc
     1381 gaggcgaaaa aatacatcgt gcagagtggg tacttcgtcg aggagaattt acagcattgg
     1441 tcttacttcc aaactaaatg ggagaactac ttgaagctgc gcggtattct tgacggtgag
     1501 tctgcgccaa ccttccctga gagctttggt gtcaaagagc gcgatcaatt ttatacatca
     1561 ttatcatatt ctggctgggg tggttcgtca ggacatgacg ctccaatgat tgcatacgat
     1621 gcagttttgg ctgcgggtga cagttggaag gagttggcgc accgcgcctt cttccacgga
     1681 ggcgattctg actccacggc cgctatcgca ggatgttggt ggggagtaat gtatggattc
     1741 aaaggggtta gtccaagcaa ctacgagaaa ttggaatacc gcaaccgctt ggaggagact
     1801 gcgcgcgctt tatatagcct gggctcgaaa gaagataccg ttattagcct ttaggacggt
     1861 ctccagtaaa ggtggatacg gatccgaatt cgagctccgt cgacaagctt gcggccgcac
     1921 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     1981 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     2041 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     2101 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2161 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2221 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2281 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     2341 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     2401 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     2461 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     2521 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     2581 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     2641 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     2701 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     2761 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     2821 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     2881 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     2941 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     3001 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     3061 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     3121 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     3181 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     3241 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     3301 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     3361 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     3421 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     3481 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     3541 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     3601 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3661 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3721 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3781 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3841 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3901 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3961 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4021 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4081 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4141 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4201 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     4261 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     4321 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     4381 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     4441 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     4501 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4561 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4621 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     4681 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     4741 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     4801 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4861 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4921 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4981 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     5041 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     5101 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     5161 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     5221 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     5281 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     5341 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     5401 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     5461 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     5521 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     5581 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5641 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     5701 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     5761 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     5821 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     5881 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     5941 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     6001 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     6061 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     6121 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6181 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6241 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     6301 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     6361 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     6421 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     6481 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     6541 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     6601 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     6661 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     6721 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     6781 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     6841 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6901 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     6961 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     7021 tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
     7081 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
     7141 gatatacat
//
