LOCUS       sequence-347414-        6576 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 6576)
  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 6576)
  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: MDO2-c005
FEATURES             Location/Qualifiers
     source          1..6576
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..21
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             25..537
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             544..564
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1353..1370
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1423..1441)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1437..1484
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1521..1976
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1608..1627)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1818..1839
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2068..2883)
                     /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(2068..2883)
                     /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     2791..2810
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2875..2894
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3005..3593
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3494..3513
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3747..3764
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3779..3918
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3844..3863)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4004..4026
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4020..4211)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLQDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5020..6102)
                     /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     6064..6083
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6103..6180)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6386..6405
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6489..6508
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6489..6507
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6508..6532
                     /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             6547..6569
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgcaccatc atcatcatca ttctatggtc ttcacactcg aagatttcgt tggggactgg
       61 cgacagacag ccggctacaa cctggaccaa gtccttgaac agggaggtgt gtccagtttg
      121 tttcagaatc tcggggtgtc cgtaactccg atccaaagga ttgtcctgag cggtgaaaat
      181 gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag gtctgagcgg cgaccaaatg
      241 ggccagatcg aaaaaatttt taaggtggtg taccctgtgg acgatcatca ctttaaggtg
      301 atcctgcact atggcacact ggtaatcgac ggggttacgc cgaacatgat cgactatttc
      361 ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa agatcactgt aacagggacc
      421 ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca accccgacgg ctccctgctg
      481 ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt gcgaacgcat tctggcgggt
      541 accgagaact tgtacttcca atccatgaag aaaaaggtgt ggagagagga gaaagaacgt
      601 ttattgaaga tgaccttaga agagagacgc aaagaatacc taagagacta tattcccctg
      661 aacagcattc tgtcatggaa ggaggagatg aagggcaagg gccaaaatga tgaagaaaat
      721 actcaggaaa catcccaggt gaagaaaagt ttgactgaaa aagtttctct ctatagaggt
      781 gacatcacat tgctagaggt agatgctata gtcaatgccg caaatgccag tcttcttgga
      841 ggaggaggtg tggatggctg tattcataga gcagccggcc cctgtttgct agctgaatgt
      901 cgtaacctga atggctgtga tactggacat gcaaaaatca catgtggcta tgaccttcct
      961 gcaaaatatg tcatccatac tgtagggcca atagccaggg gccatattaa tggttcccac
     1021 aaggaagacc ttgcaaattg ctataaatca tctctgaagc tcgtgaaaga aaataacatc
     1081 cgatcagttg catttccctg catctcaaca ggcatttatg gctttcccaa cgagcctgct
     1141 gcagtcattg ccctcaacac cattaaggaa tggcttgcca agaatcacca tgaggtggat
     1201 cggatcattt tctgtgtctt cttagaagtt gacttcaaaa tctacaaaaa gaaaatgaat
     1261 gagtttttct ccgtagacta agacggtctc cagtaaaggt ggatacggat ccgaattcga
     1321 gctccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg
     1381 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     1441 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     1501 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1561 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1621 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1681 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1741 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1801 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1861 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1921 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     1981 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     2041 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg
     2101 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     2161 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     2221 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     2281 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     2341 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     2401 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     2461 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     2521 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     2581 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     2641 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     2701 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     2761 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     2821 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
     2881 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
     2941 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3001 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3061 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3121 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3181 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3241 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3301 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3361 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3421 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3481 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3541 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3601 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3661 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3721 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3781 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     3841 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3901 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3961 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4021 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4081 tggtcctgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4141 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4201 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4261 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4321 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4381 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4441 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4501 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4561 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4621 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     4681 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     4741 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     4801 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     4861 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     4921 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     4981 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5041 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5101 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     5161 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     5221 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     5281 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     5341 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     5401 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     5461 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     5521 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     5581 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     5641 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     5701 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     5761 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     5821 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     5881 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     5941 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     6001 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     6061 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     6121 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     6181 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     6241 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     6301 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     6361 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     6421 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
     6481 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
     6541 aataattttg tttaacttta agaaggagat atacat
//
