LOCUS       sequence-417055-        5823 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 5823)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 5823)
  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; aliases: Enterovirus D68 2...
FEATURES             Location/Qualifiers
     source          1..5823
                     /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)."
     CDS             626..643
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             688..705
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(758..776)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      772..819
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      856..1311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(943..962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1153..1174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1403..2218)
                     /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(1403..2218)
                     /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     2126..2145
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2210..2229
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2340..2928
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2829..2848
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3082..3099
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3114..3253
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3179..3198)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3339..3361
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3355..3546)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4355..5437)
                     /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     5399..5418
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5438..5515)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5721..5740
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaacct taagaaggag atatacacat gaatgcgatt atagggaacc gggctagcgt
      121 gacaactatg ccgcataata ttgtcacgac gggtccgggc tttgggggcg tatttgttgg
      181 aagttttaaa attattaatt accaccttgc aacgattgag gagcgtcagt ctgcaattta
      241 cgtcgattgg cagagcgacg ttcttgttac ccctatagca gcacacggga gacaccagat
      301 tgcccggtgt aagtgtaaca cgggagttta ttactgtaga catcgcgacc gcagttatcc
      361 aatttgtttt gagggacctg gaatccagtg gattgaacaa aatgaatact atcctgcacg
      421 ttatcaaaca aatgtgttat tggccgcggg cccggcggag gctggagatt gtggcggttt
      481 actggtttgt ccccacggcg ttataggcct gttaacggcg gggggtggag gtatcgtggc
      541 gtttacagac atacggaatc tgctgtggct ggatacggat gtcatggaac agggcataac
      601 tgattatatt caaaaccttg gaaaccacca tcatcaccac cattaatgac tcgagcaccc
      661 gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct
      721 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
      781 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
      841 ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
      901 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
      961 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     1021 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     1081 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     1141 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     1201 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     1261 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     1321 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     1381 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt
     1441 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag
     1501 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga
     1561 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg
     1621 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt
     1681 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca
     1741 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag
     1801 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa
     1861 tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg
     1921 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag
     1981 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc
     2041 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga
     2101 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat
     2161 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa
     2221 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct
     2281 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     2341 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     2401 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     2461 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     2521 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     2581 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     2641 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     2701 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     2761 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     2821 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     2881 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     2941 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3001 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     3061 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     3121 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     3181 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     3241 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     3301 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     3361 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     3421 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     3481 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     3541 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     3601 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     3661 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     3721 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     3781 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     3841 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     3901 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     3961 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc
     4021 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa
     4081 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc
     4141 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac
     4201 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca
     4261 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta
     4321 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     4381 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     4441 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca
     4501 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa
     4561 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt
     4621 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg
     4681 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca
     4741 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta
     4801 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg
     4861 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat
     4921 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct
     4981 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg
     5041 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat
     5101 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc
     5161 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca
     5221 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg
     5281 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt
     5341 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg
     5401 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct
     5461 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga
     5521 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg
     5581 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     5641 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     5701 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     5761 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga
     5821 aat
//
