LOCUS       sequence-419426-        5832 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 5832)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 5832)
  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-tag, self cleaving
FEATURES             Location/Qualifiers
     source          1..5832
                     /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             887..904
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             949..966
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1019..1037)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1033..1080
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1117..1572
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1204..1223)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1414..1435
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1664..2479)
                     /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(1664..2479)
                     /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     2387..2406
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2471..2490
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2601..3189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3090..3109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3343..3360
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3375..3514
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3440..3459)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3600..3622
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3616..3807)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4616..5698)
                     /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     5660..5679
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5699..5776)
                     /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 atgaattata aaggaaatga
      361 tatcaagtgt acatcaacat cccgtgataa aataacgacc ttgggaaagt tcgggcaaca
      421 atcgggtgcc atatatgtgg gtaactatcg tgtggttaat cgccacctcg ctactcacaa
      481 tgattgggcc aacttagtgt gggaggacag ctctcgcgat ctccttgtat catcgaccac
      541 tgcgcagggt tgcgacacca ttgcgcgctg tgattgccag acaggcgtgt attattgctc
      601 gtctcgtcgt aaacattatc cggtttcttt ttctaagccg tcactgatct ttgttgaagc
      661 gtcagagtac tatccagccc gttatcagtc ccatctgatg ctggctgtgg gtcacagcga
      721 acccggggat tgtggcggta tactgcgctg tcagcacggt gtagttggca ttgtcagtac
      781 aggaggtaat gggttagtcg gatttgccga tgtgcgggat ttactgtggt tggacgagga
      841 ggctatggaa cagggcgtat ctgattatat caaaggactg ggcgaccacc atcatcacca
      901 ccattaatga ctcgagcacc cgtcgacaag cttgcggccg cactcgagca ccaccaccac
      961 caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1021 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1081 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1141 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1201 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1261 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1321 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1381 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1441 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1501 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1561 cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     1621 tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga
     1681 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     1741 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     1801 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     1861 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     1921 gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     1981 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     2041 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     2101 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     2161 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     2221 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     2281 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     2341 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     2401 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt
     2461 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     2521 ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2581 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2641 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2701 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2761 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2821 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2881 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2941 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3001 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3061 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3121 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3181 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3241 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3301 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3361 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3421 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3481 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     3541 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3601 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     3661 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3721 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3781 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3841 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     3901 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     3961 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4021 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4081 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4141 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4201 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4261 catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt
     4321 ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag
     4381 cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag
     4441 cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac
     4501 gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat
     4561 cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact
     4621 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4681 ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg
     4741 gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc
     4801 tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg
     4861 agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg
     4921 actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag
     4981 tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc
     5041 agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc
     5101 cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct
     5161 ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa
     5221 taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc
     5281 aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac
     5341 tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt
     5401 ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga
     5461 caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact
     5521 gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg
     5581 cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa
     5641 cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat
     5701 tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc
     5761 gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag
     5821 cagcccagta gt
//
