LOCUS       sequence-416946-        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: Fusion of West Nile NS2B-NS3 proteins
FEATURES             Location/Qualifiers
     source          1..6165
                     /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)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             92..109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(125..148,149..184,185..208)
                     /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             233..253
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1030..1047
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1100..1118)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1114..1161
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1198..1653
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1285..1304)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1495..1516
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1745..2560)
                     /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(1745..2560)
                     /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     2468..2487
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2552..2571
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2682..3270
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3171..3190
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3424..3441
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3456..3595
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3521..3540)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3681..3703
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3697..3888)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4697..5779)
                     /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     5741..5760
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5780..5857)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6063..6082
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atatacatat gcaccatcat catcatcatt cttctggtgc
      121 gagctggagt cacccacaat tcgagaaagg tggtggctct ggtggcggtt caggcggctc
      181 cgcctggtcc catccacagt ttgaaaaggg ttctggtgta gatctgggta ccgagaacct
      241 gtacttccaa tccatgagca ctgatatgtg gattgaacgc acggcagaca tcagctggga
      301 gtcggatgct gagatcacag gttcaagcga aagagtggat gtacgattag acgatgacgg
      361 taattttcaa ctgatgaatg accctggcgc gccgtgggca ggcggcggcg gctctggtgg
      421 aggaggtggc gttttgtggg acacgccgtc ccccaaagaa tacaagaagg gtgacacgac
      481 taccggtgtt tacagaatta tgacccgggg tttgcttggg tcgtaccagg ccggggcagg
      541 ggtgatggta gagggagttt tccatacctt atggcacact accaaaggtg ccgcacttat
      601 gtctggtgag ggtcgccttg atccttattg gggtagcgta aaagaggatc ggctctgtta
      661 cggcggacca tggaagcttc aacacaagtg gaacggacag gatgaagttc agatgattgt
      721 cgtagagccg ggcaagaatg tcaagaacgt gcaaacaaag cccggtgtat tcaaaacgcc
      781 ggaaggcgaa attggcgctg tgacccttga ctttccaact gggaccagcg gctcaccaat
      841 agtcgacaag aatggagatg tcattgggtt gtacggaaat ggtgttatta tgcccaatgg
      901 ctcgtacatc tccgctatcg tacaaggaga gcgcatggac gaaccgatac ctgcaggctt
      961 cgaacctgag atgttaagaa agaagtaatg actcgagcac ccgtcgacaa gcttgcggcc
     1021 gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa
     1081 gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa
     1141 cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg
     1201 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1261 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     1321 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     1381 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat
     1441 cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac
     1501 tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag
     1561 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
     1621 cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg
     1681 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     1741 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     1801 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     1861 tccataggat ggcaagatcc tggtatcggt ctgctattcc gactcgtcca acatcaatac
     1921 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     1981 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     2041 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     2101 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     2161 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag
     2221 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     2281 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     2341 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     2401 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     2461 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     2521 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     2581 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     2641 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2701 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2761 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2821 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2881 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2941 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     3001 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     3061 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     3121 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     3181 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     3241 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     3301 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     3361 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3421 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     3481 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     3541 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     3601 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3661 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3721 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     3781 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     3841 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     3901 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     3961 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     4021 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     4081 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     4141 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     4201 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     4261 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     4321 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     4381 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     4441 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagtggtc
     4501 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     4561 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     4621 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     4681 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4741 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     4801 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     4861 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     4921 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     4981 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     5041 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     5101 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     5161 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     5221 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     5281 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     5341 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     5401 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     5461 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     5521 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5581 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     5641 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     5701 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     5761 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     5821 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     5881 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     5941 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     6001 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     6061 tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc
     6121 acgatgcgtc cggcgtagag gatcgagatc tcgatcccgc gaaat
//
