LOCUS       sequence-416947-        6159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of a fusion of Dengue2 NS2B-NS3 proteins.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6159)
  TITLE     The SARS-Cov-2 main protease (Mpro) is responsible for viral 
            polyprotein processing and therefore a therapeutic target.
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: Fusion of Dengue2 NS2B-NS3
FEATURES             Location/Qualifiers
     source          1..6159
                     /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             1024..1041
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1094..1112)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1108..1155
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1192..1647
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1279..1298)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1489..1510
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1739..2554)
                     /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(1739..2554)
                     /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     2462..2481
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2546..2565
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2676..3264
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3165..3184
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3418..3435
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3450..3589
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3515..3534)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3675..3697
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3691..3882)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4691..5773)
                     /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     5735..5754
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5774..5851)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6057..6076
                     /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 tccatggctg atctggaatt agaacgagct gcggatgtac gctgggaaga
      301 gcaggccgag atctcgggtt ctagccccat tctttctata accatctccg aagacggtag
      361 catgtcaatc aaaaatgaag aagaggaaca gacactgggt ggtggaggat caggaggagg
      421 aggtgctggg gtgctctggg atgttccctc ccctcctccg gtaggtaaag cagagctgga
      481 agatggcgct taccggataa aacaaaaagg gattctgggt tactcgcaga ttggagcagg
      541 agtttacaag gagggtactt tccacacgat gtggcatgtt actcgtggtg cggtccttat
      601 gcataaaggt aaacgcatcg aaccgtcatg ggcagatgtc aaaaaagacc ttattagtta
      661 tggtggagga tggaagctgg aaggagaatg gaaagaaggc gaagaggttc aagtgctggc
      721 actggaacct ggtaagaatc cgcgtgcggt acagactaaa cctggcttat tcaaaacaaa
      781 taccgggaca ataggttgcg tttccctcga tttctcccca gggacctccg gctcaccaat
      841 tgttgataaa aaaggtaaag tagttggtct ttatggaaat ggggtagtca cccgaagtgg
      901 tgcatatgtc tctgccatag ccaacaccga aaaatcaatt gaggacaatc cggaaattga
      961 agacgatata ttccgcaaat aatgactcga gcacccgtcg acaagcttgc ggccgcactc
     1021 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
     1081 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
     1141 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
     1201 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     1261 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     1321 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     1381 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     1441 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     1501 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     1561 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     1621 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
     1681 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
     1741 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
     1801 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
     1861 ggatggcaag atcctggtat cggtctgcta ttccgactcg tccaacatca atacaaccta
     1921 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
     1981 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
     2041 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
     2101 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
     2161 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
     2221 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
     2281 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
     2341 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
     2401 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
     2461 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
     2521 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
     2581 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
     2641 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     2701 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     2761 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     2821 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     2881 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     2941 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     3001 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     3061 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     3121 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     3181 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     3241 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     3301 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     3361 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     3421 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
     3481 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3541 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
     3601 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
     3661 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
     3721 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
     3781 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
     3841 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
     3901 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
     3961 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
     4021 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
     4081 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
     4141 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
     4201 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
     4261 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
     4321 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
     4381 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
     4441 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagt ggtcctcgcc
     4501 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
     4561 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
     4621 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
     4681 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4741 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
     4801 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
     4861 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
     4921 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
     4981 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
     5041 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
     5101 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
     5161 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
     5221 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
     5281 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
     5341 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
     5401 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
     5461 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
     5521 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
     5581 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
     5641 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
     5701 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
     5761 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
     5821 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
     5881 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
     5941 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
     6001 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
     6061 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
     6121 cgtccggcgt agaggatcga gatctcgatc ccgcgaaat
//
