LOCUS       sequence-423081-        6438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expresison of Enterovirus A71 2A protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6438)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6438)
  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"
COMMENT     Sequence Label: Enterovirus A71 2A protease
FEATURES             Location/Qualifiers
     source          1..6438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1287..1306
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1390..1409
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1390..1408
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1409..1433
                     /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             1448..1470
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1481..1498
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1505..2158
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             2174..2194
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             2692..2709
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2762..2780)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2776..2823
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2860..3315
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2947..2966)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3157..3178
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3407..4222)
                     /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(3407..4222)
                     /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     4130..4149
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4214..4233
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4344..4932
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4833..4852
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5086..5103
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5118..5257
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5183..5202)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5343..5365
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5359..5550)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6359..6438,1..1003))
                     /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 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     1081 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     1141 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     1201 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     1261 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     1321 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     1381 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
     1441 aaataatttt gtttaacttt aagaaggaga tatacatatg caccatcacc accatcactc
     1501 atcgatgtct cccattttgg gttattggaa gataaagggg ttagtacaac cgaccagatt
     1561 actgttagag taccttgaag agaagtatga agagcattta tatgagcgcg acgaaggcga
     1621 taagtggcgc aacaagaaat ttgagttagg cctcgagttt ccgaaccttc cttattatat
     1681 cgacggggac gtaaagttga cgcaatctat ggcaataatt cggtatatag cagacaagca
     1741 taacatgttg ggtggctgcc ccaaagagcg tgccgagatc tccatgttag aaggagccgt
     1801 cctggacatt cgctatggtg tgtcgcgcat tgcctactca aaagatttcg agactttaaa
     1861 agttgacttt ctgagcaagc tgccggagat gcttaagatg ttcgaagatc ggctgtgtca
     1921 caagacgtac ctcaacggtg accacgttac ccacccagac tttatgcttt acgatgcgct
     1981 ggacgttgtg ctttacatgg acccgatgtg tctggacgcg tttccgaagt tggtatgttt
     2041 caagaagcgg attgaggcta ttccccaaat agacaaatac cttaagagta gtaagtacat
     2101 cgcgtggcca cttcaaggtt ggcaagctac cttcggcggc ggcgaccatc ccccaaagtc
     2161 gggcggcggc agcgagaacc tgtacttcca atccatggga aagttcgggc aacaatcggg
     2221 tgccatatat gtgggtaact atcgtgtggt taatcgccac ctcgctactc acaatgattg
     2281 ggccaactta gtgtgggagg acagctctcg cgatctcctt gtatcatcga ccactgcgca
     2341 gggttgcgac accattgcgc gctgtgattg ccagacaggc gtgtattatt gctcgtctcg
     2401 tcgtaaacat tatccggttt ctttttctaa gccgtcactg atctttgttg aagcgtcaga
     2461 gtactatcca gcccgttatc agtcccatct gatgctggct gtgggtcaca gcgaacccgg
     2521 ggattgtggc ggtatactgc gctgtcagca cggtgtagtt ggcattgtca gtacaggagg
     2581 taatgggtta gtcggatttg ccgatgtgcg ggatttactg tggttggacg aggaggctat
     2641 ggaacagtaa tgactcgagc acccgtcgac aagcttgcgg ccgcactcga gcaccaccac
     2701 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
     2761 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     2821 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     2881 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     2941 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3001 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3061 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3121 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3181 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3241 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     3301 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3361 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     3421 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     3481 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     3541 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     3601 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     3661 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     3721 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     3781 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     3841 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     3901 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     3961 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     4021 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     4081 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     4141 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     4201 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     4261 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4321 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4381 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4441 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     4501 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4561 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4621 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4681 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4741 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     4801 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     4861 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     4921 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     4981 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5041 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     5101 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5161 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     5221 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     5281 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     5341 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     5401 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     5461 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     5521 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     5581 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     5641 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     5701 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     5761 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     5821 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     5881 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     5941 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     6001 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     6061 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     6121 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagtgg tcctcgccga aaatgaccca
     6181 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     6241 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     6301 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     6361 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6421 cgcggggaga ggcggttt
//
