LOCUS       sequence-347411-        6396 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 6396)
  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 6396)
  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-c003
FEATURES             Location/Qualifiers
     source          1..6396
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1292..1311
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1414..1438
                     /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             1453..1475
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1486..1503
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1513..1839
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             1846..1866
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2655..2672
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2725..2743)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2739..2786
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2823..3278
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2910..2929)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3120..3141
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3370..4185)
                     /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(3370..4185)
                     /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     4093..4112
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4177..4196
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4307..4895
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4796..4815
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5049..5066
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5081..5220
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5146..5165)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5306..5328
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5322..5513)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6322..6396,1..1008))
                     /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"
ORIGIN
        1 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1201 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1261 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     1321 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     1381 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     1441 tctagaaata attttgttta actttaagaa ggagatatac atatgcacca tcatcatcat
     1501 cattcttctg gtatgagcga taaaattatt cacctgactg acgacagttt tgacacggat
     1561 gtactcaaag cggacggggc gatcctcgtc gatttctggg cagagtggtg cggtccgtgc
     1621 aaaatgatcg ccccgattct ggatgaaatc gctgacgaat atcagggcaa actgaccgtt
     1681 gcaaaactga acatcgatca aaaccctggc actgcgccga aatatggcat ccgtggtatc
     1741 ccgactctgc tgctgttcaa aaacggtgaa gtggcggcaa ccaaagtggg cgcactgtct
     1801 aaaggtcagt tgaaagagtt cctcgacgct aacctggccg gtaccgagaa cctgtacttc
     1861 caatccatga agaaaaaggt gtggagagag gagaaagaac gtttattgaa gatgacctta
     1921 gaagagagac gcaaagaata cctaagagac tatattcccc tgaacagcat tctgtcatgg
     1981 aaggaggaga tgaagggcaa gggccaaaat gatgaagaaa atactcagga aacatcccag
     2041 gtgaagaaaa gtttgactga aaaagtttct ctctatagag gtgacatcac attgctagag
     2101 gtagatgcta tagtcaatgc cgcaaatgcc agtcttcttg gaggaggagg tgtggatggc
     2161 tgtattcata gagcagccgg cccctgtttg ctagctgaat gtcgtaacct gaatggctgt
     2221 gatactggac atgcaaaaat cacatgtggc tatgaccttc ctgcaaaata tgtcatccat
     2281 actgtagggc caatagccag gggccatatt aatggttccc acaaggaaga ccttgcaaat
     2341 tgctataaat catctctgaa gctcgtgaaa gaaaataaca tccgatcagt tgcatttccc
     2401 tgcatctcaa caggcattta tggctttccc aacgagcctg ctgcagtcat tgccctcaac
     2461 accattaagg aatggcttgc caagaatcac catgaggtgg atcggatcat tttctgtgtc
     2521 ttcttagaag ttgacttcaa aatctacaaa aagaaaatga atgagttttt ctccgtagac
     2581 taagacggtc tccagtaaag gtggatacgg atccgaattc gagctccgtc gacaagcttg
     2641 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     2701 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     2761 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     2821 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2881 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2941 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3001 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3061 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3121 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3181 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3241 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     3301 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     3361 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     3421 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     3481 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     3541 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     3601 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     3661 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     3721 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     3781 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     3841 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     3901 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     3961 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     4021 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     4081 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     4141 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     4201 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     4261 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     4321 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     4381 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     4441 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     4501 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     4561 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     4621 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     4681 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     4741 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     4801 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4861 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4921 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4981 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     5041 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     5101 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     5161 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     5221 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     5281 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     5341 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     5401 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     5461 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     5521 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     5581 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     5641 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     5701 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     5761 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     5821 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     5881 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     5941 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     6001 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     6061 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     6121 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     6181 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     6241 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     6301 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     6361 ctgcattaat gaatcggcca acgcgcgggg agaggc
//
