LOCUS       sequence-416943-        6042 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 6042)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6042)
  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; no tag, low expression levels
FEATURES             Location/Qualifiers
     source          1..6042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     77..96
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     180..199
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        180..198
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    199..223
                     /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             280..297
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1086..1103
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1156..1174)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1170..1217
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1254..1709
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1341..1360)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1551..1572
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1801..2616)
                     /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(1801..2616)
                     /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     2524..2543
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2608..2627
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2738..3326
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3227..3246
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3480..3497
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3512..3651
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3577..3596)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3737..3759
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3753..3944)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4753..5835)
                     /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     5797..5816
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5836..5913)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
       61 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
      121 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
      181 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
      241 gtttaacctt aagaaggaga tatacacatg ggctcctcac atcatcacca ccaccacatg
      301 gcgagcatga gtgacagtga ggtaaaccaa gaggccaagc ccgaggtcaa gccggaagta
      361 aaacccgaga cacacatcaa cttaaaagta tctgacggtt cttctgagat attcttcaag
      421 attaagaaga cgactccact gcggagatta atggaagcat tcgcaaaacg gcaaggaaag
      481 gaaatggact ctcttcgctt cctctacgac ggcattcgta ttcaagccga tcaaaccccc
      541 gaggaccttg acatggaaga taacgacatc atcgaggcgc accgggaaca aatcggcggt
      601 ggccctggat tcggtggcgt gttcgttggg agtttcaaaa taataaatta ccaccttgca
      661 accatagagg aacgccagtc cgcgatttat gtcgattggc aatcggacgt actggtgacc
      721 cctatagctg cgcatgggcg acatcagata gctagatgta aatgcaatac cggcgtatat
      781 tattgtcgcc accgggaccg ttcgtatccc atctgtttcg aagggccagg gattcaatgg
      841 atcgagcaaa atgaatacta ccccgcgcgg tatcaaacta acgtcttatt ggcggccgga
      901 ccggcagaag ctggcgactg tggtggattg ctcgtgtgcc ctcatggggt gataggtttg
      961 ttgactgcag gaggaggtgg catagttgcg ttcacggaca taagaaactt gctctggtta
     1021 gacactgacg tgatggaaca gtaatgactc gagcacccgt cgacaagctt gcggccgcac
     1081 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     1141 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1201 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     1261 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1321 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1381 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1441 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     1501 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1561 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     1621 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1681 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     1741 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     1801 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     1861 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     1921 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     1981 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     2041 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     2101 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     2161 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     2221 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     2281 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     2341 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     2401 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     2461 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     2521 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     2581 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     2641 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     2701 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2761 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2821 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2881 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2941 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3001 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3061 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3121 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3181 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3241 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3301 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3361 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3421 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3481 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3541 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     3601 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3661 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3721 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3781 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     3841 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     3901 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     3961 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4021 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4081 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4141 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     4201 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4261 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     4321 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4381 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     4441 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     4501 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     4561 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     4621 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4681 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4741 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4801 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     4861 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     4921 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     4981 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     5041 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     5101 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     5161 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     5221 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     5281 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     5341 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     5401 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     5461 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     5521 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     5581 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     5641 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5701 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5761 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     5821 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     5881 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     5941 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     6001 ggaatggtgc atgcaaggag atggcgccca acagtccccc gg
//
