LOCUS       sequence-461586-        6123 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the fusion of the NS2B-NS3 protease from 
            Usutu virus for biochemical assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6123)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6123)
  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: USUV strain Vienna 2001 NS2B-NS3 protease fusi...
FEATURES             Location/Qualifiers
     source          1..6123
                     /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             1017..1034
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1087..1105)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1101..1148
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1185..1640
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1272..1291)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1482..1503
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1732..2547)
                     /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(1732..2547)
                     /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     2455..2474
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2539..2558
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2669..3257
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3158..3177
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3411..3428
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3443..3582
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3508..3527)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3668..3690
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3684..3875)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4684..5766)
                     /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     5728..5747
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5767..5844)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6050..6069
                     /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 ccatgagtac agatctgtgg
      301 ttggaaagag ccgcagatat cacttgggag acagacgccg caataacagg aacttcccaa
      361 cgtctggatg tcaaattgga tgatgacggt gatttccatt taattaacga tccgggggtg
      421 ccgtgggcag ggggcggcgg ctctggagga ggtggaggcg tattttggga caccccagca
      481 ccacgaacat atccgaaggg cgatacatca ccgggcgttt atcgcatcat gagccggtac
      541 atcctgggaa cttatcaggc cggcgtcggg gtgatgtatg aaggcgttct tcacacttta
      601 tggcatacaa cacgaggggc tgcgataaga agcggcgagg gccggcttac accttactgg
      661 gggagcgtaa aagaagatcg catcacgtac gggggtccgt ggaagtttga tcgtaaatgg
      721 aacggtttag acgacgtgca acttataata gttgcgccgg ggaaagctgc gataaacata
      781 cagacgaaac caggcatttt taagactcca caaggtgaga ttggagcagt tagcctggat
      841 taccctgaag gaacttcagg atctccaatc ttagataaaa atggagacat tgtaggactc
      901 tatggtaatg gagtcatttt aggcaatggt agctacgtta gcgcaattgt ccagggcgag
      961 cgtgaagagg agtaatgact cgagcacccg tcgacaagct tgcggccgca ctcgagcacc
     1021 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1081 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1141 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
     1201 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     1261 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1321 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1381 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1441 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1501 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1561 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1621 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1681 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa
     1741 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
     1801 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc
     1861 aagatcctgg tatcggtctg ctattccgac tcgtccaaca tcaatacaac ctattaattt
     1921 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
     1981 tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg
     2041 ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc
     2101 gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg
     2161 gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa
     2221 tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt
     2281 acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac
     2341 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
     2401 cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg
     2461 agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca
     2521 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
     2581 cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2641 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2701 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2761 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     2821 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2881 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2941 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     3001 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     3061 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     3121 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     3181 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     3241 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3301 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3361 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3421 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     3481 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     3541 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     3601 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3661 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     3721 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     3781 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     3841 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     3901 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     3961 gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg
     4021 cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta
     4081 ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag
     4141 ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt
     4201 gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt
     4261 gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg
     4321 agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg
     4381 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4441 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agtggtcctc gccgaaaatg
     4501 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     4561 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4621 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4681 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4741 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     4801 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     4861 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     4921 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     4981 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     5041 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     5101 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     5161 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5221 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5281 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5341 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5401 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5461 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5521 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5581 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5641 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5701 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     5761 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     5821 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     5881 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     5941 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
     6001 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     6061 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     6121 cgt
//
