LOCUS       sequence-349898-        6192 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 6192)
  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 6192)
  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: CoV-2-NSP3-c002
FEATURES             Location/Qualifiers
     source          1..6192
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..21
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             31..357
                     /codon_start=1
                     /gene="trxA"
                     /product="E. coli thioredoxin"
                     /label=TrxA
                     /translation="MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDE
                     IADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFL
                     DANLA"
     CDS             364..384
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             969..986
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1039..1057)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1053..1100
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1137..1592
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1224..1243)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1434..1455
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1684..2499)
                     /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(1684..2499)
                     /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     2407..2426
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2491..2510
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2621..3209
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3110..3129
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3363..3380
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3395..3534
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3460..3479)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3620..3642
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3636..3827)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4636..5718)
                     /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     5680..5699
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5719..5796)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6002..6021
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6105..6124
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6105..6123
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6124..6148
                     /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             6163..6185
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgcaccatc atcatcatca ttcttctggt atgagcgata aaattattca cctgactgac
       61 gacagttttg acacggatgt actcaaagcg gacggggcga tcctcgtcga tttctgggca
      121 gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg atgaaatcgc tgacgaatat
      181 cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa accctggcac tgcgccgaaa
      241 tatggcatcc gtggtatccc gactctgctg ctgttcaaaa acggtgaagt ggcggcaacc
      301 aaagtgggcg cactgtctaa aggtcagttg aaagagttcc tcgacgctaa cctggccggt
      361 accgagaact tgtacttcca atccatggag gtgaatagtt tctcagggta tttaaagttg
      421 actgataatg tttacatcaa gaacgcggat attgttgaag aggccaagaa ggtgaaacct
      481 accgtggtgg taaacgctgc aaacgtgtac cttaagcacg gcgggggagt cgcgggagca
      541 ctgaataaag ctacgaataa tgcaatgcaa gtggaatcag atgactacat cgcaaccaat
      601 ggcccattaa aagtgggtgg tagctgcgtc ctgtctggac ataacctggc aaaacactgt
      661 ttacatgtag ttgggcccaa cgtcaataaa ggagaggaca tccaattatt gaaatctgct
      721 tatgaaaact tcaatcagca cgaggtgtta cttgcaccgt tgttgtccgc tgggattttt
      781 ggcgctgacc ccattcattc tcttcgtgtc tgtgttgata ctgtgcgtac taacgtatac
      841 ctggcagttt ttgataagaa tttatatgac aaattggtgt catcttttct tgagtaggac
      901 ggtctccagt aaaggtggat acggatccga attcgagctc cgtcgacaag cttgcggccg
      961 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     1021 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     1081 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     1141 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     1201 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     1261 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     1321 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     1381 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     1441 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     1501 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     1561 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     1621 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa
     1681 ttcttagaaa aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc
     1741 aataccatat ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt
     1801 ccataggatg gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca
     1861 acctattaat ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac
     1921 gactgaatcc ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg
     1981 ccagccatta cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga
     2041 ttgcgcctga gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat
     2101 cgaatgcaac cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg
     2161 atattcttct aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc
     2221 atcatcagga gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca
     2281 gtttagtctg accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag
     2341 aaacaactct ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc
     2401 gacattatcg cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg
     2461 cggcctagag caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt
     2521 tatgtaagca gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2581 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2641 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2701 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2761 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2821 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2881 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     2941 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3001 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3061 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3121 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3181 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3241 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3301 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3361 gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc
     3421 atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc
     3481 atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga
     3541 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
     3601 agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg
     3661 aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg
     3721 tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt
     3781 ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg
     3841 taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg
     3901 atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg
     3961 gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt
     4021 aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac
     4081 ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag
     4141 accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc
     4201 tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc
     4261 ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc
     4321 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
     4381 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
     4441 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
     4501 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
     4561 ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca
     4621 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4681 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt
     4741 tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag
     4801 ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt
     4861 taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc
     4921 cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc
     4981 gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg
     5041 aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg
     5101 agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc
     5161 cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt
     5221 accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa
     5281 taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg
     5341 atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca
     5401 ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc
     5461 gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc
     5521 aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta
     5581 attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc
     5641 ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta
     5701 taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc
     5761 cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat
     5821 gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg
     5881 caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca
     5941 ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat
     6001 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
     6061 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
     6121 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
     6181 ggagatatac at
//
