LOCUS       sequence-414877-        6900 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 6900)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6900)
  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; 6xHis-MBP-TEV tag, self cl...
FEATURES             Location/Qualifiers
     source          1..6900
                     /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             1430..1447
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2579..2599
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             3097..3114
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3167..3185)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3181..3228
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3265..3720
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3352..3371)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3562..3583
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3812..4627)
                     /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(3812..4627)
                     /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     4535..4554
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4619..4638
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4749..5337
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5238..5257
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5491..5508
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5523..5662
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5588..5607)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5748..5770
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5764..5955)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6764..6900,1..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 atgaagatcc accatcatca
     1441 tcatcacgaa gagggtaagc tggtgatctg gatcaatggg gataaggggt acaacggcct
     1501 ggcagaggtc ggtaagaagt ttgagaagga cacagggatt aaggtgactg ttgagcaccc
     1561 tgataagctg gaagaaaagt tcccgcaggt tgcagccacc ggagacggcc ctgacatcat
     1621 tttctgggca cacgaccgct tcggtgggta cgcgcaatct ggactgttgg ctgaaattac
     1681 gcccgacaag gcattccaag acaaattata ccctttcacg tgggatgctg tccggtataa
     1741 cgggaagctg atagcgtatc cgattgcggt agaggcgttg tcattaatct ataacaagga
     1801 tttattaccc aatccgccca aaacttggga agagatccca gcgttggaca aggagttgaa
     1861 ggcaaagggt aaaagcgccc tcatgttcaa ccttcaagaa ccgtatttta cgtggccatt
     1921 aatcgccgct gacggtgggt acgcattcaa gtacgagaac ggaaagtacg atatcaagga
     1981 tgttggggtt gacaacgctg gggccaaagc gggcttaacg ttcctcgttg acctcataaa
     2041 gaacaagcac atgaacgcgg atacggacta ctcgatagct gaagctgctt ttaacaaagg
     2101 cgagactgct atgaccatta acggaccttg ggcgtggtcc aacattgaca catcaaaggt
     2161 gaactacggg gttacagtac ttccgacctt taaagggcaa cctagcaagc cattcgtggg
     2221 agtcctgtca gccggaatta atgcggcgtc accgaacaag gagttagcga aggagtttct
     2281 tgagaactat ttacttaccg acgagggctt ggaagccgtt aacaaggaca agccacttgg
     2341 ggcggtagca ttaaaatcgt acgaagaaga attggcgaag gacccgcgga tcgccgcgac
     2401 gatggaaaat gcccagaagg gcgagattat gccgaacatt ccccaaatgt ctgcattctg
     2461 gtatgcagtt cgcacggcag tgataaacgc agccagcggt cgtcaaacgg tggacgaggc
     2521 tcttaaggat gcccaaacga actccataac atcgctgtac aagaaggctg gatctggtga
     2581 gaacctgtac ttccaatcca tgggaaagtt cgggcaacaa tcgggtgcca tatatgtggg
     2641 taactatcgt gtggttaatc gccacctcgc tactcacaat gattgggcca acttagtgtg
     2701 ggaggacagc tctcgcgatc tccttgtatc atcgaccact gcgcagggtt gcgacaccat
     2761 tgcgcgctgt gattgccaga caggcgtgta ttattgctcg tctcgtcgta aacattatcc
     2821 ggtttctttt tctaagccgt cactgatctt tgttgaagcg tcagagtact atccagcccg
     2881 ttatcagtcc catctgatgc tggctgtggg tcacagcgaa cccggggatt gtggcggtat
     2941 actgcgctgt cagcacggtg tagttggcat tgtcagtaca ggaggtaatg ggttagtcgg
     3001 atttgccgat gtgcgggatt tactgtggtt ggacgaggag gctatggaac agtaatgact
     3061 cgagcacccg tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
     3121 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     3181 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     3241 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3301 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3361 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3421 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3481 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3541 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3601 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3661 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3721 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     3781 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     3841 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     3901 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     3961 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     4021 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     4081 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     4141 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     4201 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     4261 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     4321 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     4381 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     4441 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     4501 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     4561 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     4621 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     4681 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4741 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4801 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4861 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     4921 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4981 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     5041 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5101 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5161 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5221 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5281 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5341 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5401 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     5461 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     5521 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     5581 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     5641 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     5701 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     5761 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     5821 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     5881 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     5941 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     6001 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     6061 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     6121 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     6181 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     6241 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     6301 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     6361 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     6421 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     6481 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     6541 catcgtcgcg ctccagcgaa agtggtcctc gccgaaaatg acccagagcg ctgccggcac
     6601 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     6661 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     6721 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     6781 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     6841 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
//
