LOCUS       sequence-419427-        5844 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expresison of Enterovirus A71 3C protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5844)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 5844)
  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: EV-A71 3C protease; aliases: Enterovirus A71 3...
FEATURES             Location/Qualifiers
     source          1..5844
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     150..169
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     253..272
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        253..271
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    272..296
                     /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)."
     CDS             899..916
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             961..978
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1031..1049)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1045..1092
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1129..1584
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1216..1235)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1426..1447
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1676..2491)
                     /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(1676..2491)
                     /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     2399..2418
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2483..2502
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2613..3201
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3102..3121
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3355..3372
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3387..3526
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3452..3471)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3612..3634
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3628..3819)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4628..5710)
                     /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     5672..5691
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5711..5788)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
       61 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
      121 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
      181 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
      241 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      301 tagaaataat tttgtttaac cttaagaagg agatatacac atgggtccta gcctggattt
      361 tgcgttgtca ttgctgcgcc gtaatatcag acaggttcag accgatcagg gccatttcac
      421 catgctcgga gttcgcgatc gtttggcggt gctcccgcgt cactctcagc caggtaaaac
      481 gatttgggtt gaacataaat taataaacat attggacgcc gttgagctgg ttgatgaaca
      541 gggtgtaaac ttagaactga ctttagtgac cctggatacc aatgaaaagt ttcgcgacat
      601 taccaaattt atccccgaaa atatctcagc cgcctcagat gccacactgg ttatcaacac
      661 tgagcacatg cccagcatgt ttgtcccggt tggagatgta gttcagtacg gttttcttaa
      721 cctgagcggt aagcctacac atcgtaccat gatgtataac tttccgacga aggctggtca
      781 gtgtgggggt gtggttacga gtgttggtaa agtgatcggg atacatatag gagggaatgg
      841 tcgtcagggc ttttgcgctg gtctgaaacg ttcgtatttt gcatcagaac agcttgagca
      901 ccatcatcac caccattaat gactcgagca cccgtcgaca agcttgcggc cgcactcgag
      961 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     1021 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1081 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1141 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1201 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1261 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1321 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1381 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1441 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1501 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1561 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1621 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     1681 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     1741 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     1801 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     1861 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     1921 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     1981 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2041 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     2101 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2161 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     2221 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     2281 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     2341 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     2401 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     2461 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     2521 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     2581 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     2641 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     2701 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     2761 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     2821 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     2881 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     2941 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3001 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3061 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3121 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3181 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3241 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     3301 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     3361 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     3421 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3481 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     3541 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     3601 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     3661 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     3721 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     3781 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     3841 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     3901 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     3961 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     4021 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     4081 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     4141 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     4201 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4261 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     4321 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     4381 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     4441 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     4501 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     4561 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     4621 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4681 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     4741 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     4801 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     4861 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     4921 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     4981 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     5041 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     5101 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     5161 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     5221 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     5281 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     5341 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     5401 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     5461 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     5521 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     5581 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     5641 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     5701 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     5761 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
     5821 tgcattagga agcagcccag tagt
//
