LOCUS       sequence-443792-        6165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a fusion of West Nile NS2B-NS3 proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6165)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6165)
  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: WNV NS2B-NS3 protease (K104A - catalytically a...
FEATURES             Location/Qualifiers
     source          1..6165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     73..92
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(112..189)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     395..414
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     498..517
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        498..516
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    517..541
                     /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             556..578
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             589..606
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(622..645,646..681,682..705)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             730..750
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1527..1544
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1597..1615)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1611..1658
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1695..2150
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1782..1801)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1992..2013
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2242..3057)
                     /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(2242..3057)
                     /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     2965..2984
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3049..3068
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3179..3767
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3668..3687
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3921..3938
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3953..4092
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4018..4037)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4178..4200
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4194..4385)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5194..6165,1..111))
                     /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 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
       61 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
      121 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
      181 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
      241 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
      301 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
      361 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
      421 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
      481 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
      541 ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgca ccatcatcat
      601 catcattctt ctggtgcgag ctggagtcac ccacaattcg agaaaggtgg tggctctggt
      661 ggcggttcag gcggctccgc ctggtcccat ccacagtttg aaaagggttc tggtgtagat
      721 ctgggtaccg agaacctgta cttccaatcc atgagcactg atatgtggat tgaacgcacg
      781 gcagacatca gctgggagtc ggatgctgag atcacaggtt caagcgaaag agtggatgta
      841 cgattagacg atgacggtaa ttttcaactg atgaatgacc ctggcgcgcc gtggaaaggc
      901 ggcggcggct ctggtggagg aggtggcgtt ttgtgggaca cgccgtcccc caaagaatac
      961 aagaagggtg acacgactac cggtgtttac agaattatga cccggggttt gcttgggtcg
     1021 taccaggccg gggcaggggt gatggtagag ggagttttcc ataccttatg gcacactacc
     1081 aaaggtgccg cacttatgtc tggtgagggt cgccttgatc cttattgggg tagcgtaaaa
     1141 gaggatcggc tctgttacgg cggaccatgg aagcttcaac acaagtggaa cggacaggat
     1201 gaagttcaga tgattgtcgt agagccgggc aagaatgtca agaacgtgca aacaaagccc
     1261 ggtgtattca aaacgccgga aggcgaaatt ggcgctgtga cccttgactt tccaactggg
     1321 accagcggct caccaatagt cgacaagaat ggagatgtca ttgggttgta cggaaatggt
     1381 gttattatgc ccaatggctc gtacatctcc gctatcgtac aaggagagcg catggacgaa
     1441 ccgatacctg caggcttcga acctgagatg ttaagaaaga agtaatgact cgagcacccg
     1501 tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta
     1561 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     1621 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     1681 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1741 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1801 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1861 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1921 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1981 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     2041 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     2101 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     2161 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2221 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2281 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2341 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg ctattccgac
     2401 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2461 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2521 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     2581 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     2641 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     2701 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     2761 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     2821 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     2881 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     2941 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3001 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     3061 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     3121 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     3181 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     3241 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     3301 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     3361 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3421 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3481 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3541 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3601 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3661 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3721 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3781 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3841 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3901 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3961 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     4021 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     4081 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     4141 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     4201 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     4261 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     4321 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     4381 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     4441 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4501 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     4561 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     4621 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4681 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4741 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4801 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     4861 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4921 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     4981 ctccagcgaa agtggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     5041 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     5101 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     5161 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     5221 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5281 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     5341 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     5401 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     5461 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     5521 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     5581 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     5641 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     5701 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5761 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5821 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5881 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     5941 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     6001 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     6061 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     6121 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccac
//
