LOCUS       sequence-461559-        6159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the fusion of the NS2B-NS3 protease from 
            Dengue virus serotype 1 for biochemical assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6159)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6159)
  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-1 strain US/Hawaii/1944 NS2B-NS3 protease...
FEATURES             Location/Qualifiers
     source          1..6159
                     /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             1053..1070
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1123..1141)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1137..1184
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1221..1676
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1308..1327)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1518..1539
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1768..2583)
                     /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(1768..2583)
                     /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     2491..2510
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2575..2594
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2705..3293
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3194..3213
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3447..3464
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3479..3618
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3544..3563)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3704..3726
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3720..3911)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4720..5802)
                     /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     5764..5783
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5803..5880)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6086..6105
                     /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 ccatggcaga cttaagtctt
      301 gagaaagccg cagaaatctc gtgggaagaa gaggcagaac attccggagc ttcccacaat
      361 atcctggtag aagtgcagga tgacggtaca atgaaaatca aagacgaaga gcgtgacgac
      421 acgggcggtg gcggttctgg cggggggggt tctggtgttt tgtgggatac tccttcgccg
      481 cctgaggttg aacgtgctgt gctggacgac ggtatttata gaatattgca acgaggtctg
      541 ctcggtcgct ctcaagtcgg agtcggtgtt ttccaagagg gtgtgttcca cacaatgtgg
      601 cacgtgacac gtggcgccgt tttaatgtac caagggaagc gcctggaacc gagctgggcc
      661 agcgtaaaga aggatttgat ctcttatggc ggcggttggc gttttcaagg aagttggaac
      721 actggggaag aagttcaggt cattgccgtt gagccaggca agaatccgaa gaatgttcaa
      781 acagccccag gaacctttaa aacaccggaa ggtgaagtgg gagctatagc cctggacttt
      841 aagcctggca catctgggtc ccccatagtc aaccgggaag gcaagattgt cggtctttat
      901 gggaacgggg tggtcactac cagcggaacc tatgtctcag caatcgcaca agccaaggcc
      961 agccaagagg ggcctttacc ggagatcgaa gatgaggtat tccgtaaata atgactcgag
     1021 cacccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg
     1081 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     1141 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     1201 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     1261 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     1321 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     1381 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     1441 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     1501 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     1561 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     1621 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     1681 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     1741 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg
     1801 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     1861 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgctat
     1921 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     1981 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     2041 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     2101 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     2161 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     2221 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     2281 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     2341 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     2401 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     2461 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     2521 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
     2581 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
     2641 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     2701 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     2761 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     2821 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     2881 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     2941 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3001 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3061 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3121 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3181 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3241 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3301 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     3361 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     3421 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     3481 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     3541 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     3601 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     3661 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     3721 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     3781 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     3841 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     3901 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     3961 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4021 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4081 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4141 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4201 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4261 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4321 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggg
     4381 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     4441 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     4501 gtcgcgctcc agcgaaagtg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     4561 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     4621 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     4681 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4741 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4801 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     4861 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     4921 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     4981 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     5041 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     5101 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     5161 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     5221 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     5281 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     5341 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     5401 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     5461 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     5521 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     5581 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     5641 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     5701 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     5761 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     5821 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     5881 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     5941 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     6001 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     6061 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     6121 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgt
//
