LOCUS       sequence-414876-        6036 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 6036)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6036)
  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 2A protease; aliases: Enterovirus EV71 ...
FEATURES             Location/Qualifiers
     source          1..6036
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     908..927
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(947..1024)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1230..1249
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1333..1352
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1333..1351
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1352..1376
                     /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             1391..1413
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1424..1441
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2233..2250
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2303..2321)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2317..2364
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2401..2856
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2488..2507)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2698..2719
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2948..3763)
                     /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(2948..3763)
                     /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     3671..3690
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3755..3774
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3885..4473
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4374..4393
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4627..4644
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4659..4798
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4724..4743)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4884..4906
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4900..5091)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5900..6036,1..946))
                     /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 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
       61 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
      121 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
      181 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
      241 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
      301 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
      361 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
      421 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
      481 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
      541 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
      601 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
      661 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
      721 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
      781 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
      841 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
      901 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
      961 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     1021 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     1081 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     1141 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     1201 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
     1261 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
     1321 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     1381 tagaaataat tttgtttaac tttaagaagg agatatacat atgcaccacc atcatcacca
     1441 tggctcagga gatcaggaag cgaaaccttc gaccgaggac ctgggcgaca agaaagaagg
     1501 ggagtatatt aagttaaagg tgatcggcca ggactcaagc gagatccact tcaaagtcaa
     1561 aatgaccacg cacctcaaga agttgaagga gagttactgt caacggcaag gggttcctat
     1621 gaactcgctt cgcttcctgt tcgagggcca gcgaattgcg gacaaccaca cacctaagga
     1681 acttggtatg gaagaggaag acgtcattga ggtgtaccag gagcagacag gcggcatggg
     1741 aaagttcggg caacaatcgg gtgccatata tgtgggtaac tatcgtgtgg ttaatcgcca
     1801 cctcgctact cacaatgatt gggccaactt agtgtgggag gacagctctc gcgatctcct
     1861 tgtatcatcg accactgcgc agggttgcga caccattgcg cgctgtgatt gccagacagg
     1921 cgtgtattat tgctcgtctc gtcgtaaaca ttatccggtt tctttttcta agccgtcact
     1981 gatctttgtt gaagcgtcag agtactatcc agcccgttat cagtcccatc tgatgctggc
     2041 tgtgggtcac agcgaacccg gggattgtgg cggtatactg cgctgtcagc acggtgtagt
     2101 tggcattgtc agtacaggag gtaatgggtt agtcggattt gccgatgtgc gggatttact
     2161 gtggttggac gaggaggcta tggaacagta atgactcgag cacccgtcga caagcttgcg
     2221 gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag
     2281 gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct
     2341 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg
     2401 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     2461 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     2521 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     2581 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     2641 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     2701 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     2761 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     2821 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     2881 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa
     2941 ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     3001 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     3061 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     3121 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     3181 tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa
     3241 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     3301 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     3361 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     3421 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     3481 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     3541 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     3601 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     3661 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     3721 atcgcggcct agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     3781 tgtttatgta agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg
     3841 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3901 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3961 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4021 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4081 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4141 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4201 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4261 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4321 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4381 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4441 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4501 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4561 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4621 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     4681 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     4741 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4801 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4861 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4921 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4981 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     5041 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     5101 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     5161 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     5221 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     5281 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     5341 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc
     5401 gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt
     5461 tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg
     5521 ggtcctcaac gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat
     5581 ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc
     5641 gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagtg
     5701 gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa
     5761 gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac
     5821 tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact
     5881 tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     5941 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg
     6001 tttttctttt caccagtgag acgggcaaca gctgat
//
