LOCUS       sequence-419428-        6114 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 6114)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
REFERENCE   2  (bases 1 to 6114)
  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 (based on PDB 2YOL,
            cata...
FEATURES             Location/Qualifiers
     source          1..6114
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     150..169
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     253..272
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        253..271
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    272..296
                     /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             311..333
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             344..361
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(377..400,401..436,437..460)
                     /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             485..505
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1231..1248
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1301..1319)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1315..1362
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1399..1854
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1486..1505)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1696..1717
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1946..2761)
                     /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(1946..2761)
                     /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     2669..2688
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2753..2772
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2883..3471
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3372..3391
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3625..3642
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3657..3796
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3722..3741)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3882..3904
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3898..4089)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4898..5980)
                     /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     5942..5961
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5981..6058)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
       61 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
      121 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
      181 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
      241 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      301 tagaaataat tttgtttaac tttaagaagg agatatacat atgcaccatc atcatcatca
      361 ttcttctggt gcgagctgga gtcacccaca attcgagaaa ggtggtggct ctggtggcgg
      421 ttcaggcggc tccgcctggt cccatccaca gtttgaaaag ggttctggtg tagatctggg
      481 taccgagaac ctgtacttcc aatccatgac tgatatgtgg attgagcgta cagcagatat
      541 aacctgggag tctgacgccg aaatcactgg ttcatccgaa cgagtagacg ttcggttaga
      601 tgacgacggt aattttcagt tgatgaatga tccaggagcc ccctgggctg gcggtggagg
      661 atcaggtggg gggggaggcg gcgtgttgtg ggatactcca tcccctaaag aatataaaaa
      721 aggagatact actacggggg tttatcgtat aatgacaaga ggtcttctgg ggtcttatca
      781 agcaggagct ggtgttatgg tagaaggagt gtttcatacc ctctggcata ccacaaaggg
      841 cgcagctttg atgtcaggag aaggtcggct ggatccatat tggggatcgg tgaaagagga
      901 tcgtttatgt tatggcggac cgtggaaact gcagcacaag tggaatggtc atgatgaagt
      961 tcaaatgatc gtagttgaac ctgggaaaaa cgtaaaaaac gtgcaaacca aaccgggcgt
     1021 gtttaaaacg cctgagggcg aaattggggc agtaacgttg gattatccca caggaacctc
     1081 cggcagcccg attgtggata agaatggaga tgttattggc ttatatggca atggagtgat
     1141 tatgccaaat ggcagttaca tatcagccat tgtgcagggt gagcgctaat gactcgagca
     1201 cccgtcgaca agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct
     1261 gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca
     1321 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     1381 tccggattgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     1441 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     1501 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     1561 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     1621 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     1681 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     1741 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     1801 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg
     1861 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1921 tatgtatccg ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca
     1981 atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag
     2041 gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgctattc
     2101 cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa
     2161 gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt
     2221 ctttccagac ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa
     2281 ccaaaccgtt attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa
     2341 aaggacaatt acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa
     2401 caatattttc acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga
     2461 tcgcagtggt gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa
     2521 gaggcataaa ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa
     2581 cgctaccttt gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat
     2641 agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag
     2701 catccatgtt ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca
     2761 taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc
     2821 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     2881 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     2941 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3001 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3061 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3121 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3181 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3241 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3301 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3361 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3421 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3481 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3541 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3601 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     3661 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     3721 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     3781 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     3841 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     3901 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     3961 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4021 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     4081 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4141 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4201 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4261 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     4321 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     4381 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     4441 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     4501 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc
     4561 cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac
     4621 gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt
     4681 cgcgctccag cgaaagtggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc
     4741 tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc
     4801 ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc
     4861 ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4921 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4981 tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
     5041 tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
     5101 gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
     5161 cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
     5221 ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
     5281 tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
     5341 ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
     5401 ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
     5461 gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
     5521 tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
     5581 tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
     5641 gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
     5701 cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
     5761 gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
     5821 gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
     5881 ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
     5941 cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac
     6001 tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg
     6061 ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag tagt
//
