LOCUS       sequence-419420-        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 proteins
FEATURES             Location/Qualifiers
     source          1..6159
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     150..169
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     253..272
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        253..271
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    272..296
                     /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             311..333
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             344..361
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(377..400,401..436,437..460)
                     /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             485..505
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1276..1293
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1346..1364)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1360..1407
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1444..1899
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1531..1550)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1741..1762
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1991..2806)
                     /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(1991..2806)
                     /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     2714..2733
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2798..2817
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2928..3516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3417..3436
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3670..3687
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3702..3841
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3767..3786)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3927..3949
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3943..4134)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4943..6025)
                     /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     5987..6006
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6026..6103)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
       61 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
      121 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
      181 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatctcg
      241 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
      301 tagaaataat tttgtttaac tttaagaagg agatatacat atgcaccatc atcatcatca
      361 ttcttctggt gcgagctgga gtcacccaca attcgagaaa ggtggtggct ctggtggcgg
      421 ttcaggcggc tccgcctggt cccatccaca gtttgaaaag ggttctggtg tagatctggg
      481 taccgagaac ctgtacttcc aatccatggc cgacctcgaa ttagaaagag ccgcagatgt
      541 caagtgggaa gatcaggccg agatctccgg ttcttcaccc atactcagca taactattag
      601 cgaggatgga tccatgtcaa tcaagaacga agaggaagag cagacactgg gcggaggagg
      661 aagcggcggt ggcggggcag gtgtattatg ggacgtccca agtccgcccc caatgggaaa
      721 agccgaactg gaagatggcg cctatcgcat aaagcaaaag ggaatcttag gctatagtca
      781 gataggggcg ggtgtgtata aggaaggtac atttcacacc atgtgggcag tgacccgcgg
      841 ggcggtactc atgcataagg gaaagcggat tgagccaagc tgggccgatg taaagaaaga
      901 tttgatttcc tatggcggtg gatggaagct cgaaggcgag tggaaagagg gcgaagaggt
      961 ccaagttctg gccctggagc ctggtaagaa cccgcgtgcg gtgcaaacca agccaggcct
     1021 gttcaagaca aacgcgggaa ctataggtgc cgtgtcgctg gacttctccc ctggcacctc
     1081 aggttccccg attatcgaca agaaggggaa agtcgttggc ttatacggaa acggtgttgt
     1141 tacgcgttct ggtgcgtacg tatccgcaat tgcccaaacg gagaagagca tcgaggataa
     1201 cccggagatc gaggacgaca ttttcagaaa ataatgactc gagcacccgt cgacaagctt
     1261 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1321 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1381 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1441 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1501 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1561 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1621 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1681 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1741 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1801 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1861 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1921 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     1981 gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag
     2041 gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga
     2101 ggcagttcca taggatggca agatcctggt atcggtctgc tattccgact cgtccaacat
     2161 caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat
     2221 gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt
     2281 caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca
     2341 ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa
     2401 caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg
     2461 aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta
     2521 accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg
     2581 tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat
     2641 gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg
     2701 attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat
     2761 ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat
     2821 tactgtttat gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt
     2881 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     2941 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     3001 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     3061 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     3121 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     3181 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     3241 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     3301 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     3361 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     3421 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     3481 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     3541 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     3601 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     3661 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     3721 cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg
     3781 atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc
     3841 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     3901 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     3961 atcaccgaaa cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc
     4021 acagatgtct gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt
     4081 ctggcttctg ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc
     4141 ctccgtgtaa gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat
     4201 gctcacgata cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa
     4261 acaactggcg gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg
     4321 cttcgttaat acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat
     4381 ccggaacata atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa
     4441 accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca
     4501 cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag
     4561 ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat
     4621 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     4681 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     4741 gtggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     4801 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     4861 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     4921 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4981 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     5041 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     5101 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     5161 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     5221 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     5281 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     5341 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     5401 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     5461 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     5521 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     5581 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     5641 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     5701 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     5761 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     5821 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     5881 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     5941 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     6001 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     6061 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     6121 cccttatgcg actcctgcat taggaagcag cccagtagt
//
