LOCUS       sequence-461602-        6153 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the fusion of the NS2B-NS3 protease from 
            Zika virus for biochemical assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6153)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6153)
  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: ZIKV isolate VE_Ganxian2016 NS2B-NS3 protease ...
FEATURES             Location/Qualifiers
     source          1..6153
                     /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             1047..1064
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1117..1135)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1131..1178
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1215..1670
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1302..1321)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1512..1533
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1762..2577)
                     /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(1762..2577)
                     /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     2485..2504
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2569..2588
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2699..3287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3188..3207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3441..3458
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3473..3612
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3538..3557)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3698..3720
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3714..3905)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4714..5796)
                     /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     5758..5777
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5797..5874)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6080..6099
                     /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 ccatgagtgg aaagtctgtc
      301 gatatgtaca tcgagcgagc cggcgacatc acatgggaga aagacgcaga ggttaccggg
      361 aatagcccgc ggcttgacgt tgcgttggac gagagtggtg acttctctct ggtcgaggac
      421 gacggaccac ctatgcgtga gggaggaggt gggtcaggtg gtggtggtgg ctcgggtgct
      481 ctttgggatg tccctgcccc caaagaggtg aagaagggtg agacgaccga cggagtgtat
      541 cgcgttatga caagacgttt attagggtca acacaagtag gtgttggtgt aatgcaagag
      601 ggcgttttcc acacaatgtg gcacgttact aagggctcag ctcttcggtc aggtgaggga
      661 cgccttgacc cgtactgggg cgacgttaag caagatctgg tgtcttattg tggtccctgg
      721 aagttagacg ctgcatggga cggtcatagc gaggtccaat tgctggcagt gcccccgggt
      781 gagagagcgc gaaatattca aacgcttcct gggattttca agaccaagga cggtgacatc
      841 ggtgcagtgg cgctggatta cccggcggga acgtccggca gtccgatttt ggataagtgc
      901 ggtcgggtaa tcggactgta tggtaatggg gtggtgatta agaacggctc atacgtgtct
      961 gcaataaccc agggccgtcg agaggaagaa actcctgttg agtaatgact cgagcacccg
     1021 tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta
     1081 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
     1141 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     1201 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     1261 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     1321 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     1381 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     1441 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     1501 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     1561 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1621 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     1681 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1741 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     1801 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     1861 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg ctattccgac
     1921 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     1981 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2041 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     2101 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     2161 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     2221 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     2281 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     2341 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     2401 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     2461 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     2521 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     2581 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     2641 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2701 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2761 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2821 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2881 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2941 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3001 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3061 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3121 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3181 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3241 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3301 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3361 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3421 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3481 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     3541 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3601 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3661 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3721 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3781 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3841 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     3901 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     3961 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4021 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     4081 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     4141 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4201 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4261 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4321 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     4381 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     4441 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     4501 ctccagcgaa agtggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     4561 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     4621 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     4681 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4741 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4801 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     4861 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     4921 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     4981 tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     5041 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     5101 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     5161 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     5221 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     5281 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     5341 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     5401 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     5461 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     5521 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     5581 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     5641 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     5701 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     5761 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     5821 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     5881 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     5941 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     6001 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     6061 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     6121 cgccggtgat gccggccacg atgcgtccgg cgt
//
