LOCUS       sequence-443794-        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 (catalytically active, s...
FEATURES             Location/Qualifiers
     source          1..6165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..77
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(97..174)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     380..399
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     483..502
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        483..501
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    502..526
                     /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             541..563
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             574..591
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(607..630,631..666,667..690)
                     /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             715..735
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1512..1529
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1582..1600)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1596..1643
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1680..2135
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1767..1786)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1977..1998
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2227..3042)
                     /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(2227..3042)
                     /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     2950..2969
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3034..3053
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3164..3752
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3653..3672
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3906..3923
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3938..4077
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4003..4022)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4163..4185
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4179..4370)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5179..6165,1..96))
                     /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 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
       61 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
      121 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
      181 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
      241 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
      301 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
      361 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
      421 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa
      481 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
      541 tttgtttaac tttaagaagg agatatacat atgcaccatc atcatcatca ttcttctggt
      601 gcgagctgga gtcacccaca attcgagaaa ggtggtggct ctggtggcgg ttcaggcggc
      661 tccgcctggt cccatccaca gtttgaaaag ggttctggtg tagatctggg taccgagaac
      721 ctgtacttcc aatccatgag cacggatatg tggatagaac gtacagccga cattagctgg
      781 gaaagtgacg ctgaaataac cggatccagt gagcgcgtcg atgtgcgctt agatgacgac
      841 ggaaatttcc agttgatgaa tgatccgggt gctccgtgga aaggtggcgg cgggtcgggt
      901 ggcggcggcg gtgttctgtg ggatacaccc tcacctaagg aatataagaa aggggacacg
      961 acaacagggg tatacagaat catgacgcgc ggtctgcttg gctcctacca ggcaggggct
     1021 ggtgtcatgg tagaaggcgt cttccacact ctctgggcta cgactaaagg ggcggctctg
     1081 atgtctggtg aaggtcggtt ggacccgtac tggggctctg taaaggaaga ccgcctctgt
     1141 tatggtgggc cttggaaatt acaacacaaa tggaacggtc aagatgaggt tcaaatgatc
     1201 gtagtggagc cgggtaagaa cgtaaagaac gttcaaacaa agccaggcgt atttaaaacc
     1261 cccgaaggcg agatcggcgc cgtcacgctg gactttccca caggtacaag cggttctcct
     1321 attgtagaca agaatggtga cgtaattggc ctctacggca atggtgtcat aatgccgaac
     1381 ggctcgtata tcagcgcaat tgtacaaggt gaacggatgg acgagcctat cccggctggt
     1441 ttcgaacctg agatgttgcg taagaagtaa tgactcgagc acccgtcgac aagcttgcgg
     1501 ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     1561 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     1621 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     1681 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     1741 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     1801 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     1861 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     1921 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     1981 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2041 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2101 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     2161 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     2221 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     2281 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     2341 gttccatagg atggcaagat cctggtatcg gtctgctatt ccgactcgtc caacatcaat
     2401 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     2461 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     2521 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     2581 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     2641 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     2701 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     2761 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     2821 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     2881 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     2941 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     3001 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     3061 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     3121 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3181 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3241 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3301 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3361 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3421 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3481 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3541 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3601 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3661 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3721 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3781 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     3841 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     3901 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     3961 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4021 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     4081 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4141 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4201 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4261 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4321 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4381 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4441 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4501 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4561 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4621 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     4681 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     4741 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     4801 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     4861 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     4921 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagtgg
     4981 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     5041 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     5101 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     5161 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5221 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     5281 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     5341 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     5401 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     5461 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     5521 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     5581 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     5641 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     5701 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5761 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5821 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5881 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5941 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     6001 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     6061 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     6121 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gtttt
//
