LOCUS       sequence-349925-        6885 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 6885)
  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 6885)
  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: MDO1-c014
FEATURES             Location/Qualifiers
     source          1..6885
                     /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             1662..1679
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1732..1750)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1746..1793
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1830..2285
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1917..1936)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2127..2148
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2377..3192)
                     /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(2377..3192)
                     /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     3100..3119
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3184..3203
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3314..3902
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3803..3822
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4056..4073
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4088..4227
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4153..4172)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4313..4335
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4329..4520)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5329..6411)
                     /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     6373..6392
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6412..6489)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6695..6714
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6798..6817
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6798..6816
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6817..6841
                     /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             6856..6878
                     /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 agaacctgta cttccaatcc
      781 atgtctgcgg gcgttggggc gtggggagcg gcagctgttg gacgtactgc aggcgtacgt
      841 acatgggctc cattagctat ggcagcaaag gtggacctta gtacgagtac agactggaag
      901 gaggccaagt cattcttaaa agggttatcc gacaagcaac gcgaggagca ttatttctgt
      961 aaagacttcg tacgtcttaa aaagattcct acatggaagg aaatggccaa gggtgtggct
     1021 gtcaaggttg aggagcctcg ctacaagaag gacaagcagt tgaatgaaaa gatcagtttg
     1081 ttacgctctg atattacaaa attagaagtc gatgctattg tcaatgcagc caatagcagt
     1141 cttttaggtg gtggaggcgt agatggatgt atccaccgtg cagcaggtcc gctgttgaca
     1201 gatgagtgtc gtactcttca gtcatgtaag acagggaagg cgaagatcac aggtggttac
     1261 cgcctgccag cgaagtatgt gatccacacg gtcggcccta ttgcctatgg agaaccctcc
     1321 gctagtcagg ctgcggagtt acgttcctgt tacttgtcct ctttggatct gttgctggaa
     1381 caccgtttac gctcagtcgc atttccatgc atttcaacgg gtgtattcgg atacccgtgc
     1441 gaggcagctg cggagatcgt tttagcaacg ttacgcgaat ggttagaaca gcacaaagat
     1501 aaagtagacc gtttaattat ttgcgtattt ttagaaaaag acgaagacat ctatcgcagt
     1561 cgcctgcccc attattttcc ggtggcttaa gacggtctcc agtaaaggtg gatacggatc
     1621 cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac caccaccact
     1681 gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     1741 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     1801 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     1861 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1921 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     1981 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2041 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2101 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2161 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     2221 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     2281 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2341 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
     2401 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
     2461 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
     2521 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
     2581 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
     2641 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
     2701 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
     2761 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     2821 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     2881 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     2941 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     3001 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     3061 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     3121 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     3181 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     3241 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     3301 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     3361 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     3421 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     3481 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     3541 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     3601 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     3661 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3721 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3781 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3841 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3901 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3961 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     4021 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     4081 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     4141 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     4201 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     4261 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     4321 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     4381 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     4441 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     4501 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     4561 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     4621 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     4681 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     4741 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     4801 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     4861 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     4921 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     4981 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     5041 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     5101 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     5161 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     5221 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     5281 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     5341 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     5401 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     5461 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     5521 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     5581 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     5641 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     5701 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     5761 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     5821 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     5881 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     5941 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6001 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6061 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6121 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     6181 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     6241 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     6301 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     6361 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     6421 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     6481 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     6541 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     6601 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     6661 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     6721 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     6781 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     6841 ccctctagaa ataattttgt ttaactttaa gaaggagata tacat
//
