LOCUS       sequence-443797-        6027 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 6027)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6027)
  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-SUMO, low expression...
FEATURES             Location/Qualifiers
     source          1..6027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     519..538
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(558..635)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     841..860
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     944..963
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        944..962
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    963..987
                     /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             1002..1024
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1035..1052
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1835..1852
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1905..1923)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1919..1966
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2003..2458
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2090..2109)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2300..2321
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2550..3365)
                     /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(2550..3365)
                     /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     3273..3292
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3357..3376
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3487..4075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3976..3995
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4229..4246
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4261..4400
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4326..4345)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4486..4508
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4502..4693)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5502..6027,1..557))
                     /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 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
       61 ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
      121 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
      181 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
      241 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
      301 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
      361 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
      421 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
      481 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
      541 aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc
      601 ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg
      661 cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc
      721 aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac
      781 catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc
      841 ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac
      901 gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata
      961 ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag
     1021 gagatataca tatgcaccac catcatcacc atggctcagg agatcaggaa gcgaaacctt
     1081 cgaccgagga cctgggcgac aagaaagaag gggagtatat taagttaaag gtgatcggcc
     1141 aggactcaag cgagatccac ttcaaagtca aaatgaccac gcacctcaag aagttgaagg
     1201 agagttactg tcaacggcaa ggggttccta tgaactcgct tcgcttcctg ttcgagggcc
     1261 agcgaattgc ggacaaccac acacctaagg aacttggtat ggaagaggaa gacgtcattg
     1321 aggtgtacca ggagcagaca ggcggcatgg gtccgggctt tgggggcgta tttgttggaa
     1381 gttttaaaat tattaattac caccttgcaa cgattgagga gcgtcagtct gcaatttacg
     1441 tcgattggca gagcgacgtt cttgttaccc ctatagcagc acacgggaga caccagattg
     1501 cccggtgtaa gtgtaacacg ggagtttatt actgtagaca tcgcgaccgc agttatccaa
     1561 tttgttttga gggacctgga atccagtgga ttgaacaaaa tgaatactat cctgcacgtt
     1621 atcaaacaaa tgtgttattg gccgcgggcc cggcggaggc tggagattgt ggcggtttac
     1681 tggtttgtcc ccacggcgtt ataggcctgt taacggcggg gggtggaggt atcgtggcgt
     1741 ttacagacat acggaatctg ctgtggctgg atacggatgt catggaacag taatgactcg
     1801 agcacccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc
     1861 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     1921 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     1981 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2041 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2101 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2161 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2221 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2281 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2341 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2401 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2461 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2521 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     2581 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     2641 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     2701 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     2761 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     2821 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     2881 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     2941 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     3001 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     3061 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     3121 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     3181 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     3241 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3301 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     3361 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     3421 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3481 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3541 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3601 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     3661 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     3721 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     3781 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3841 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3901 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3961 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4021 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4081 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4141 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4201 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4261 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     4321 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     4381 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4441 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4501 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     4561 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     4621 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     4681 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     4741 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     4801 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     4861 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4921 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4981 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     5041 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     5101 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     5161 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     5221 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5281 tcgtcgcgct ccagcgaaag tggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5341 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5401 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5461 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5521 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5581 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     5641 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     5701 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     5761 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     5821 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     5881 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     5941 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6001 cgcgccgaga cagaacttaa tgggccc
//
