LOCUS       sequence-414763-        6051 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 6051)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6051)
  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; no tag, self cleaving
FEATURES             Location/Qualifiers
     source          1..6051
                     /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)."
     CDS             1490..1507
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2305..2322
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2375..2393)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2389..2436
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2473..2928
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2560..2579)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2770..2791
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3020..3835)
                     /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(3020..3835)
                     /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     3743..3762
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3827..3846
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3957..4545
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4446..4465
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4699..4716
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4731..4870
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4796..4815)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4956..4978
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4972..5163)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5972..6051,1..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 gtttaacctt aagaaggaga tatacacatg ggctcgagcc accaccacca
     1501 tcaccacatg gcttccatgt cagacagcga ggtgaatcaa gaagccaaac cagaagtaaa
     1561 gcctgaagtc aagcccgaaa cgcatattaa tttgaaagtt agtgatggct cctctgagat
     1621 cttcttcaag ataaagaaga cgactccgtt gagaagattg atggaagcat tcgcaaagag
     1681 acaaggaaag gagatggatt cattgcgctt cctgtacgac gggattcgga tacaggctga
     1741 ccaaacaccg gaagacttgg atatggaaga caatgatatc atagaagcac atagagaaca
     1801 gattggtggc gggaaatttg gtcaacaatc cggcgccatc tacgtaggga attaccgggt
     1861 cgtgaaccgc catctggcga cccacaatga ctgggctaac ttggtctggg aagacagcag
     1921 tcgtgattta ctcgttagct caacaaccgc acaaggctgt gacaccatcg cccgctgcga
     1981 ttgccagacc ggtgtttatt actgttcctc acgtcgtaag cactatcctg tttcgtttag
     2041 caagccatct ctgatattcg tggaagcgag cgaatactat ccagcgagat accaatctca
     2101 tctgatgtta gcggtgggcc attcagagcc cggcgattgt ggcggcatat tacggtgcca
     2161 gcatggagtt gtcggtattg tttctacagg tggtaatggc ttagtcggat ttgccgacgt
     2221 cagagactta ttatggttag atgaagaggc aatggagcaa taatgactcg agcacccgtc
     2281 gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac
     2341 aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc
     2401 cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga
     2461 ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2521 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2581 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2641 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2701 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     2761 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     2821 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     2881 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     2941 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3001 tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat
     3061 tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     3121 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     3181 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     3241 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc
     3301 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     3361 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     3421 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     3481 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     3541 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     3601 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     3661 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     3721 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     3781 tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac
     3841 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa
     3901 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3961 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4021 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4081 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4141 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4201 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4261 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4321 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4381 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4441 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4501 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4561 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4621 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4681 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     4741 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     4801 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4861 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4921 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4981 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     5041 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     5101 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     5161 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     5221 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     5281 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     5341 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     5401 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga
     5461 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     5521 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     5581 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat
     5641 gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc
     5701 ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct
     5761 ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag
     5821 ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg
     5881 gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg
     5941 agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     6001 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt t
//
