LOCUS       sequence-461561-        6150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the fusion of the NS2B-NS3 protease from 
            Dengue virus serotype 3 for biochemical assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6150)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6150)
  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: DENV-3 strain Phillippines/H87/1956 NS2B-NS3
            p...
FEATURES             Location/Qualifiers
     source          1..6150
                     /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             1044..1061
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1114..1132)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1128..1175
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1212..1667
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1299..1318)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1509..1530
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1759..2574)
                     /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(1759..2574)
                     /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     2482..2501
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2566..2585
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2696..3284
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3185..3204
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3438..3455
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3470..3609
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3535..3554)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3695..3717
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3711..3902)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4711..5793)
                     /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     5755..5774
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5794..5871)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6077..6096
                     /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 ccatggctga tttgacagtg
      301 gaaaaggcgg ctgacgtaac gtgggaagag gaagccgagc aaaccggagt tagccataat
      361 ttgatgatta cggtagatga cgatggaacg atgagaatta aagatgacga gactgagaac
      421 atattaggcg gcggcggtag cggggggggt ggatcaggag tgctctggga tgtccccagc
      481 ccgccggaaa cacaaaaggc tgaactggaa gagggagtct atcgcatcaa acagcaagga
      541 atatttggaa agacacaggt tggcgtcggt gtgcaaaagg aaggcgtttt ccatacaatg
      601 tggcacgtaa cgcggggcgc agtacttacc cacaatggca agcggttaga gcccaattgg
      661 gcgtctgtca agaaggactt gatttcctac ggtggtggct ggcgcttgag cgcgcaatgg
      721 caaaagggtg aggaagtcca ggtgattgcg gttgagcctg gcaagaatcc aaagaacttt
      781 caaactatgc ctggaatctt ccaaaccacc actggtgaaa tcggcgctat cgcactggac
      841 tttaagcctg ggacttcagg ttcccccata atcaaccggg aaggaaaggt tgttgggctg
      901 tatggcaacg gcgtagtgac caagaacggt ggatacgtta gcggcattgc tcaaacaaat
      961 gccgagcccg atggcccgac gcccgagctt gaggaagaat aatgactcga gcacccgtcg
     1021 acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
     1081 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     1141 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     1201 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1261 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1321 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1381 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1441 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1501 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1561 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1621 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     1681 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1741 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     1801 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     1861 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcta ttccgactcg
     1921 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     1981 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     2041 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     2101 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     2161 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     2221 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     2281 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     2341 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     2401 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     2461 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     2521 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     2581 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     2641 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2701 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2761 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     2821 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     2881 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     2941 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3001 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3061 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3121 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3181 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3241 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3301 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3361 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3421 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3481 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     3541 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3601 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3661 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3721 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga
     3781 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     3841 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     3901 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     3961 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     4021 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     4081 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     4141 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     4201 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     4261 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4321 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     4381 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     4441 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     4501 cagcgaaagt ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     4561 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     4621 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4681 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4741 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4801 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     4861 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     4921 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     4981 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     5041 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     5101 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     5161 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     5221 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     5281 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5341 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5401 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5461 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5521 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5581 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5641 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5701 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5761 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     5821 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     5881 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     5941 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     6001 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     6061 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     6121 cggtgatgcc ggccacgatg cgtccggcgt
//
