LOCUS       sequence-461601-        6165 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the fusion of the NS2B-NS3 protease from 
            West Nile virus with an H163A - catalytically inactive mutation for 
            xxxxx.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6165)
  TITLE     ASAP Discovery Plasmids 2024
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 strain-NY99 NS2B-NS3 protease fusion with ...
FEATURES             Location/Qualifiers
     source          1..6165
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        30..48
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    49..73
                     /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             88..110
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             121..138
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(154..177,178..213,214..237)
                     /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             262..282
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1059..1076
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1129..1147)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1143..1190
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1227..1682
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1314..1333)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1524..1545
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1774..2589)
                     /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(1774..2589)
                     /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     2497..2516
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2581..2600
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2711..3299
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3200..3219
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3453..3470
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3485..3624
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3550..3569)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3710..3732
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3726..3917)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4726..5808)
                     /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     5770..5789
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5809..5886)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6092..6111
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
       61 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg
      121 caccatcatc atcatcattc ttctggtgcg agctggagtc acccacaatt cgagaaaggt
      181 ggtggctctg gtggcggttc aggcggctcc gcctggtccc atccacagtt tgaaaagggt
      241 tctggtgtag atctgggtac cgagaacctg tacttccaat ccatgagcac tgatatgtgg
      301 attgaacgca cggcagacat cagctgggag tcggatgctg agatcacagg ttcaagcgaa
      361 agagtggatg tacgattaga cgatgacggt aattttcaac tgatgaatga ccctggcgcg
      421 ccgtgggcag gcggcggcgg ctctggtgga ggaggtggcg ttttgtggga cacgccgtcc
      481 cccaaagaat acaagaaggg tgacacgact accggtgttt acagaattat gacccggggt
      541 ttgcttgggt cgtaccaggc cggggcaggg gtgatggtag agggagtttt ccatacctta
      601 tggcacacta ccaaaggtgc cgcacttatg tctggtgagg gtcgccttga tccttattgg
      661 ggtagcgtaa aagaggatcg gctctgttac ggcggaccat ggaagcttca acacaagtgg
      721 aacggacagg atgaagttca gatgattgtc gtagagccgg gcaagaatgt caagaacgtg
      781 caaacaaagc ccggtgtatt caaaacgccg gaaggcgaaa ttggcgctgt gacccttgac
      841 tttccaactg ggaccagcgg ctcaccaata gtcgacaaga atggagatgt cattgggttg
      901 tacggaaatg gtgttattat gcccaatggc tcgtacatct ccgctatcgt acaaggagag
      961 cgcatggacg aaccgatacc tgcaggcttc gaacctgaga tgttaagaaa gaagtaatga
     1021 ctcgagcacc cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag
     1081 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     1141 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     1201 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     1261 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     1321 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     1381 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     1441 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     1501 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     1561 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     1621 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     1681 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     1741 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     1801 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     1861 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     1921 tgctattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     1981 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     2041 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     2101 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     2161 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     2221 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     2281 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     2341 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     2401 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     2461 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     2521 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     2581 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     2641 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2701 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2761 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2821 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     2881 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2941 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3001 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3061 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3121 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3181 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3241 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3301 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3361 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3421 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     3481 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     3541 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     3601 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     3661 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     3721 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     3781 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     3841 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     3901 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     3961 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     4021 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4081 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4141 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4201 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4261 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4321 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4381 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     4441 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     4501 atcatcgtcg cgctccagcg aaagtggtcc tcgccgaaaa tgacccagag cgctgccggc
     4561 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     4621 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     4681 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     4741 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4801 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     4861 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     4921 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     4981 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     5041 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5101 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5161 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5221 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5281 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5341 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     5401 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     5461 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     5521 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     5581 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     5641 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     5701 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     5761 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     5821 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     5881 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     5941 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6001 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6061 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     6121 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgt
//
