LOCUS       sequence-349931-        7053 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 7053)
  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 7053)
  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: Gi-c009
FEATURES             Location/Qualifiers
     source          1..7053
                     /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 YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE
                     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             1830..1847
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1900..1918)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1914..1961
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1998..2453
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2085..2104)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2295..2316
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2545..3360)
                     /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(2545..3360)
                     /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     3268..3287
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3352..3371
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3482..4070
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3971..3990
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4224..4241
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4256..4395
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4321..4340)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4481..4503
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4497..4688)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5497..6579)
                     /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     6541..6560
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6580..6657)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6863..6882
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6966..6985
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6966..6984
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6985..7009
                     /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             7024..7046
                     /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 ggccacaagt tcagcgtgtc cggcgagggc
      121 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      181 cccgtgccct ggcccaccct cgtgaccacc ttcggctacg gcctgatgtg cttcgcccgc
      241 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      301 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      361 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
      421 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
      481 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
      541 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
      601 ctgctgcccg acaaccacta cctgagctac cagtccaagc tgagcaaaga ccccaacgag
      661 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
      721 gacgagctgt acaagtcttc tggtgtagat ctgggtaccg agaacctgta cttccaatcc
      781 atggcgcgtg aagtgaagct gctgctgctg ggtgcgggtg aaagcggcaa gagcaccatt
      841 gttaagcaga tgaaaatcat tcacgaagcg ggctacagcg aggaagagtg caagcagtac
      901 aaagcggtgg tttatagcaa caccatccaa agcatcattg cgatcattcg tgcgatgggt
      961 cgtctgaaaa ttgactttgg tgatagcgcg cgtgcggatg atgcgcgtca actgttcgtt
     1021 ctggcgggtg cggcggaaga gggctttatg accgcggaac tggcgggtgt gatcaagcgt
     1081 ctgtggaaag acagcggcgt tcaggcgtgc tttaaccgta gccgtgagta ccaactgaac
     1141 gatagcgcgg cgtactatct gaacgacctg gatcgtatcg cgcagccgaa ctatattccg
     1201 acccagcaag acgtgctgcg tacccgtgtt aagaccaccg gtatcgtgga aacccacttc
     1261 acctttaagg acctgcactt caaaatgttt gatgttggtg gccaacgtag cgagcgtaag
     1321 aaatggattc actgcttcga gggcgtgacc gcgatcatct tctgcgttgc gctgagcgac
     1381 tacgatctgg tgctggcgga agacgaagag atgaaccgta tgcacgagag catgaagctg
     1441 ttcgacagca tctgcaacaa caaatggttt accgatacca gcatcattct gttcctgaac
     1501 aagaaggacc tgttcgaaga gaaaatcaag aaaagcccgc tgaccatttg ctacccggag
     1561 tatgcgggta gcaacaccta cgaagaggcg gcggcgtata tccagtgcca attcgaagac
     1621 ctgaacaagc gtaaagatac caaggagatt tatacccact ttacctgcgc gaccgacacc
     1681 aaaaacgtgc agttcgtttt tgacgcggtg accgatgtta tcatcaagaa caacctgaag
     1741 gattgcggcc tgttctaaga cggtctccag taaaggtgga tacggatccg aattcgagct
     1801 ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
     1861 ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat
     1921 aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat
     1981 ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     2041 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     2101 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     2161 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     2221 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     2281 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     2341 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     2401 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     2461 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     2521 atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat gaaactgcaa
     2581 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     2641 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     2701 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     2761 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     2821 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     2881 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     2941 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     3001 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt tcccggggat
     3061 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     3121 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     3181 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaatcgata
     3241 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     3301 atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa tatggctcat
     3361 aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg accaaaatcc
     3421 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     3481 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     3541 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     3601 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     3661 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     3721 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     3781 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     3841 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     3901 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     3961 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     4021 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     4081 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     4141 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     4201 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
     4261 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
     4321 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
     4381 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     4441 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     4501 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
     4561 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
     4621 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
     4681 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
     4741 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
     4801 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
     4861 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     4921 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     4981 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     5041 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     5101 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac ccgtggggcc
     5161 gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg
     5221 aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc gatcatcgtc
     5281 gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct
     5341 acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat gccccgcgcc
     5401 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     5461 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5521 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5581 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt
     5641 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg
     5701 aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc
     5761 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat
     5821 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt
     5881 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc
     5941 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc
     6001 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag
     6061 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt
     6121 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat
     6181 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag
     6241 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac
     6301 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg
     6361 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg
     6421 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt
     6481 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc
     6541 ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact
     6601 ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg
     6661 gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga
     6721 ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc
     6781 ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt
     6841 ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg
     6901 tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc
     6961 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
     7021 aattttgttt aactttaaga aggagatata cat
//
