LOCUS       sequence-417056-        6429 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 6429)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6429)
  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-GST-TEV, low express...
FEATURES             Location/Qualifiers
     source          1..6429
                     /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             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             116..769
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             785..805
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1294..1311
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1364..1382)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1378..1425
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1462..1917
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1549..1568)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1759..1780
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2009..2824)
                     /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(2009..2824)
                     /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     2732..2751
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2816..2835
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2946..3534
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3435..3454
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3688..3705
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3720..3859
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3785..3804)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3945..3967
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3961..4152)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4961..6043)
                     /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     6005..6024
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6044..6121)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6327..6346
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcac caccatcact catcgatgtc
      121 tcccattttg ggttattgga agataaaggg gttagtacaa ccgaccagat tactgttaga
      181 gtaccttgaa gagaagtatg aagagcattt atatgagcgc gacgaaggcg ataagtggcg
      241 caacaagaaa tttgagttag gcctcgagtt tccgaacctt ccttattata tcgacgggga
      301 cgtaaagttg acgcaatcta tggcaataat tcggtatata gcagacaagc ataacatgtt
      361 gggtggctgc cccaaagagc gtgccgagat ctccatgtta gaaggagccg tcctggacat
      421 tcgctatggt gtgtcgcgca ttgcctactc aaaagatttc gagactttaa aagttgactt
      481 tctgagcaag ctgccggaga tgcttaagat gttcgaagat cggctgtgtc acaagacgta
      541 cctcaacggt gaccacgtta cccacccaga ctttatgctt tacgatgcgc tggacgttgt
      601 gctttacatg gacccgatgt gtctggacgc gtttccgaag ttggtatgtt tcaagaagcg
      661 gattgaggct attccccaaa tagacaaata ccttaagagt agtaagtaca tcgcgtggcc
      721 acttcaaggt tggcaagcta ccttcggcgg cggcgaccat cccccaaagt cgggcggcgg
      781 cagcgagaac ctgtacttcc aatccatggg tccgggcttt gggggcgtat ttgttggaag
      841 ttttaaaatt attaattacc accttgcaac gattgaggag cgtcagtctg caatttacgt
      901 cgattggcag agcgacgttc ttgttacccc tatagcagca cacgggagac accagattgc
      961 ccggtgtaag tgtaacacgg gagtttatta ctgtagacat cgcgaccgca gttatccaat
     1021 ttgttttgag ggacctggaa tccagtggat tgaacaaaat gaatactatc ctgcacgtta
     1081 tcaaacaaat gtgttattgg ccgcgggccc ggcggaggct ggagattgtg gcggtttact
     1141 ggtttgtccc cacggcgtta taggcctgtt aacggcgggg ggtggaggta tcgtggcgtt
     1201 tacagacata cggaatctgc tgtggctgga tacggatgtc atggaacagt aatgactcga
     1261 gcacccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg
     1321 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1381 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1441 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1501 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1561 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1621 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1681 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1741 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1801 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1861 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     1921 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1981 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     2041 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     2101 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     2161 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     2221 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     2281 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     2341 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     2401 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2461 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     2521 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     2581 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     2641 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     2701 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     2761 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     2821 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     2881 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2941 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3001 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3061 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3121 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3181 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3241 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3301 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3361 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3421 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3481 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3541 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3601 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3661 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3721 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     3781 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     3841 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3901 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3961 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     4021 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     4081 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     4141 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     4201 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     4261 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     4321 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4381 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4441 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4501 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4561 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4621 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     4681 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     4741 cgtcgcgctc cagcgaaagt ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     4801 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     4861 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     4921 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4981 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     5041 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     5101 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     5161 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     5221 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     5281 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     5341 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5401 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5461 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5521 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     5581 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     5641 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     5701 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     5761 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     5821 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     5881 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     5941 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     6001 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     6061 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     6121 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     6181 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     6241 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     6301 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     6361 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
     6421 ccgcgaaat
//
