LOCUS       sequence-414762-        5841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expresison of Enterovirus D68 3C protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5841)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 5841)
  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-D68 3C protease; aliases: Enterovirus A71 3...
FEATURES             Location/Qualifiers
     source          1..5841
                     /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             2033..2050
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2095..2112
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2165..2183)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2179..2226
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2263..2718
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2350..2369)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2560..2581
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2810..3625)
                     /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(2810..3625)
                     /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     3533..3552
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3617..3636
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3747..4335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4236..4255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4489..4506
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4521..4660
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4586..4605)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4746..4768
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4762..4953)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5762..5841,1..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 ggaccgggat tcgacttcgc
     1501 acaagctatt atgaaaaaaa atacggtgat cgcgcggacc gagaaaggcg agtttaccat
     1561 gctgggggta tatgaccgcg tggccgttat ccctacacac gcttctgttg gcgaaattat
     1621 ttacataaat gatgtagaaa cgcgcgtttt ggatgcgtgt gccttaagag acctgaccga
     1681 cacaaatctc gaaattacaa tagtgaagct ggatcgaaac cagaagttta gagacattcg
     1741 tcatttcctg cccagatgtg aagacgacta taacgatgcg gttttgtccg tgcatactag
     1801 taagtttccc aacatgtata ttccagtggg ccaggtgacc aattatggtt ttcttaacct
     1861 tgggggaact ccgacgcatc gtatcttgat gtataatttt ccaactagag cgggccagtg
     1921 cggtggtgtg gtcacaacta caggtaaagt aatcggcatt cacgttggtg ggaatggggc
     1981 tcagggtttt gcagcgatgc tgctgcactc ctatttcacc gatacccaaa agcaccatca
     2041 tcaccaccat taatgactcg agcacccgtc gacaagcttg cggccgcact cgagcaccac
     2101 caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct
     2161 gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     2221 tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg
     2281 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2341 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2401 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2461 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2521 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2581 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     2641 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2701 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2761 tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact
     2821 catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt
     2881 gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa
     2941 gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc
     3001 cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg
     3061 agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct
     3121 cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga
     3181 gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc
     3241 gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata
     3301 cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac
     3361 ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca
     3421 tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg
     3481 catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag
     3541 cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag
     3601 acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca
     3661 gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     3721 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3781 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3841 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     3901 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3961 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4021 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4081 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4141 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4201 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4261 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4321 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4381 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4441 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     4501 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     4561 tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     4621 atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc
     4681 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     4741 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca
     4801 gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc
     4861 cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc
     4921 catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct
     4981 gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga
     5041 tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg
     5101 gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg
     5161 tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg
     5221 cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt
     5281 tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga
     5341 ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag
     5401 cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa
     5461 acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac
     5521 cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac
     5581 ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     5641 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5701 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     5761 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5821 acgcgcgggg agaggcggtt t
//
