LOCUS       sequence-417053-        6891 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expresison of Enterovirus D68 2A protease.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6891)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6891)
  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 2A protease; 6xHis-MBP-TEV tag, low
            exp...
FEATURES             Location/Qualifiers
     source          1..6891
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             98..115
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1247..1267
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1756..1773
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1826..1844)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1840..1887
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1924..2379
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2011..2030)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2221..2242
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2471..3286)
                     /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(2471..3286)
                     /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     3194..3213
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3278..3297
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3408..3996
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3897..3916
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4150..4167
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4182..4321
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4247..4266)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4407..4429
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4423..4614)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5423..6505)
                     /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     6467..6486
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6506..6583)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6789..6808
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gaagatccac catcatcatc atcacgaaga
      121 gggtaagctg gtgatctgga tcaatgggga taaggggtac aacggcctgg cagaggtcgg
      181 taagaagttt gagaaggaca cagggattaa ggtgactgtt gagcaccctg ataagctgga
      241 agaaaagttc ccgcaggttg cagccaccgg agacggccct gacatcattt tctgggcaca
      301 cgaccgcttc ggtgggtacg cgcaatctgg actgttggct gaaattacgc ccgacaaggc
      361 attccaagac aaattatacc ctttcacgtg ggatgctgtc cggtataacg ggaagctgat
      421 agcgtatccg attgcggtag aggcgttgtc attaatctat aacaaggatt tattacccaa
      481 tccgcccaaa acttgggaag agatcccagc gttggacaag gagttgaagg caaagggtaa
      541 aagcgccctc atgttcaacc ttcaagaacc gtattttacg tggccattaa tcgccgctga
      601 cggtgggtac gcattcaagt acgagaacgg aaagtacgat atcaaggatg ttggggttga
      661 caacgctggg gccaaagcgg gcttaacgtt cctcgttgac ctcataaaga acaagcacat
      721 gaacgcggat acggactact cgatagctga agctgctttt aacaaaggcg agactgctat
      781 gaccattaac ggaccttggg cgtggtccaa cattgacaca tcaaaggtga actacggggt
      841 tacagtactt ccgaccttta aagggcaacc tagcaagcca ttcgtgggag tcctgtcagc
      901 cggaattaat gcggcgtcac cgaacaagga gttagcgaag gagtttcttg agaactattt
      961 acttaccgac gagggcttgg aagccgttaa caaggacaag ccacttgggg cggtagcatt
     1021 aaaatcgtac gaagaagaat tggcgaagga cccgcggatc gccgcgacga tggaaaatgc
     1081 ccagaagggc gagattatgc cgaacattcc ccaaatgtct gcattctggt atgcagttcg
     1141 cacggcagtg ataaacgcag ccagcggtcg tcaaacggtg gacgaggctc ttaaggatgc
     1201 ccaaacgaac tccataacat cgctgtacaa gaaggctgga tctggtgaga acctgtactt
     1261 ccaatccatg ggtccgggct ttgggggcgt atttgttgga agttttaaaa ttattaatta
     1321 ccaccttgca acgattgagg agcgtcagtc tgcaatttac gtcgattggc agagcgacgt
     1381 tcttgttacc cctatagcag cacacgggag acaccagatt gcccggtgta agtgtaacac
     1441 gggagtttat tactgtagac atcgcgaccg cagttatcca atttgttttg agggacctgg
     1501 aatccagtgg attgaacaaa atgaatacta tcctgcacgt tatcaaacaa atgtgttatt
     1561 ggccgcgggc ccggcggagg ctggagattg tggcggttta ctggtttgtc cccacggcgt
     1621 tataggcctg ttaacggcgg ggggtggagg tatcgtggcg tttacagaca tacggaatct
     1681 gctgtggctg gatacggatg tcatggaaca gtaatgactc gagcacccgt cgacaagctt
     1741 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1801 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1861 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1921 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1981 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2041 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     2101 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2161 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2221 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     2281 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     2341 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     2401 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2461 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     2521 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     2581 ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat
     2641 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     2701 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     2761 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     2821 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     2881 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     2941 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     3001 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     3061 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     3121 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     3181 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     3241 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     3301 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     3361 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     3421 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3481 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3541 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3601 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3661 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3721 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3781 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3841 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3901 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3961 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     4021 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     4081 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     4141 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     4201 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     4261 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     4321 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     4381 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     4441 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     4501 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     4561 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     4621 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     4681 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4741 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4801 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4861 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4921 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4981 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     5041 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     5101 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     5161 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     5221 gtggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     5281 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     5341 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     5401 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     5461 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     5521 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     5581 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     5641 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     5701 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     5761 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5821 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     5881 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     5941 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     6001 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     6061 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     6121 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     6181 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     6241 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     6301 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     6361 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     6421 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     6481 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     6541 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     6601 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     6661 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     6721 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     6781 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     6841 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa t
//
