LOCUS       sequence-461582-        6147 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the fusion of the NS2B-NS3 protease from 
            Spondweni virus for biochemical assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6147)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6147)
  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: SPOV strain SM-6 V-1 NS2B-NS3 protease fusion ...
FEATURES             Location/Qualifiers
     source          1..6147
                     /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             1041..1058
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1111..1129)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1125..1172
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1209..1664
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1296..1315)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1506..1527
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1756..2571)
                     /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(1756..2571)
                     /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     2479..2498
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2563..2582
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2693..3281
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3182..3201
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3435..3452
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3467..3606
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3532..3551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3692..3714
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3708..3899)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4708..5790)
                     /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     5752..5771
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5791..5868)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6074..6093
                     /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 ccatgtcggg gaagtcggtt
      301 gatatgtata tcgaaaaggt ctgtgacata tcatgggaca aggacgcaga gatcacgggg
      361 acgtcacctc gccttgacgt agccttggat gacagtggag acttctctct tatccaagat
      421 gatggcccac caacgcgcga gggcggcggt ggcagcggtg gtggtggtgg tgcaatgtgg
      481 gacattccta gcccgagaga ggtgaagaag ggtgaaacaa ccgctggcgt ctatcgtatt
      541 atgacacgta agctgttagg ttcaacccaa gtaggggctg gagtcatgca tgagggtgtc
      601 ttccacacga tgtggcacgt aacaaagggg tccgcactcc gctcgggcga gggacgtctg
      661 gatccgtatt ggggcaacgt aaagcaagat ctgatctcat actgcggacc gtggaagttg
      721 gacgggaaat gggacggcgt tagcgaggta caactgatag ctgtagcccc tggcgagcgt
      781 gcccgtaatg tccaaaccaa accaggtgtg tttaagacga ccgatggtga gatcggagcc
      841 cttgccttag attttccggg cggatcgagt gggagtccaa ttatcgacaa gaacgggcat
      901 gttatcggct tatacggtaa cggggtcgtt gttaaaagtg gttcttatgt aagtgcgata
      961 atgcaaacgg agaagatgga agagcccgca gtggattaat gactcgagca cccgtcgaca
     1021 agcttgcggc cgcactcgag caccaccacc accaccactg agatccggct gctaacaaag
     1081 cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg
     1141 gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggattgg
     1201 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     1261 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     1321 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     1381 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     1441 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     1501 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     1561 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc ttatttaaca
     1621 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
     1681 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1741 ctcatgaatt aattcttaga aaaactcatc gagcatcaaa tgaaactgca atttattcat
     1801 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     1861 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgctattc cgactcgtcc
     1921 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     1981 accatgagtg acgactgaat ccggtgagaa tggcaaaagt ttatgcattt ctttccagac
     2041 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt
     2101 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgctgttaa aaggacaatt
     2161 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     2221 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt
     2281 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     2341 ttccgtcagc cagtttagtc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     2401 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     2461 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     2521 ggaatttaat cgcggcctag agcaagacgt ttcccgttga atatggctca taacacccct
     2581 tgtattactg tttatgtaag cagacagttt tattgttcat gaccaaaatc ccttaacgtg
     2641 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     2701 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     2761 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     2821 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact
     2881 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     2941 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     3001 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     3061 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     3121 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     3181 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     3241 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     3301 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     3361 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     3421 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt
     3481 ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg
     3541 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     3601 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     3661 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     3721 ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc
     3781 gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt
     3841 aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact
     3901 gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag
     3961 aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag
     4021 ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc
     4081 cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg
     4141 cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca
     4201 cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg
     4261 cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag
     4321 cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg
     4381 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4441 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4501 cgaaagtggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4561 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     4621 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     4681 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4741 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4801 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     4861 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     4921 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     4981 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     5041 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5101 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5161 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5221 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5281 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5341 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5401 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5461 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5521 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     5581 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     5641 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     5701 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     5761 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     5821 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     5881 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     5941 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
     6001 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
     6061 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
     6121 tgatgccggc cacgatgcgt ccggcgt
//
