LOCUS       sequence-461584-        6126 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the fusion of the NS2B-NS3 protease from St.
            Louis encephalitis virus for biochemical assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6126)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6126)
  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: SLEV strain Kern217 NS2B-NS3 protease fusion w...
FEATURES             Location/Qualifiers
     source          1..6126
                     /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             1020..1037
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1090..1108)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1104..1151
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1188..1643
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1275..1294)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1485..1506
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1735..2550)
                     /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(1735..2550)
                     /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     2458..2477
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2542..2561
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2672..3260
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3161..3180
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3414..3431
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3446..3585
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3511..3530)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3671..3693
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3687..3878)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4687..5769)
                     /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     5731..5750
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5770..5847)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6053..6072
                     /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 ccatgagtgg taaagccgct
      301 gaaatgtgga ttgaaaaggc ggcagatatt acttgggaac agaatgcaga gataacgggc
      361 acctctccga gactcgatgt agacctggac agccacggca actttaaact gctgaatgat
      421 cctggtgctc cagggggtgg tgggtctggg ggcggagggg gcgcactctg ggatgttccc
      481 tctcctaaag tatatcctaa gtgcgaaact aaacccggta tttaccgtat tatgactcgc
      541 gggatcttag gaacgttcca agccggtgta ggagttatgc acgagggagt tttccacacc
      601 atgtggcacg cgacggaagg tgccgtctta cggaatggag aaggtcgact ggatccttac
      661 gcgggagacg tacggaatga tctgatttct tatggggggc cttggaagct gagcgctact
      721 tgggacggca cagaggaagt ccagatgatt gccgtggctc ccggcaagcc ggcgataaat
      781 gttcaaacta cgcccggggt attcaagaca cctctgggca caatcggcgc tgttacttta
      841 gattttccga aagggacctc gggatcaccc ataatcaata aaaaagggga aattataggg
      901 ctgtacggaa atggcgtcct tatcggccag ggggagtacg tgtcgggaat tattcaggga
      961 gaacgcaccg aggaataatg actcgagcac ccgtcgacaa gcttgcggcc gcactcgagc
     1021 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     1081 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     1141 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     1201 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1261 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1321 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1381 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     1441 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1501 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     1561 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1621 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     1681 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa
     1741 aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata
     1801 tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat
     1861 ggcaagatcc tggtatcggt ctgctattcc gactcgtcca acatcaatac aacctattaa
     1921 tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc
     1981 cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt
     2041 acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg
     2101 agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa
     2161 ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc
     2221 taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg
     2281 agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct
     2341 gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc
     2401 tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc
     2461 gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga
     2521 gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc
     2581 agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2641 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2701 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2761 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2821 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2881 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     2941 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3001 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3061 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3121 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3181 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3241 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3301 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3361 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3421 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3481 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3541 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3601 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3661 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3721 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3781 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3841 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3901 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     3961 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     4021 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     4081 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     4141 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     4201 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     4261 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     4321 aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg
     4381 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     4441 aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagtggtc ctcgccgaaa
     4501 atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata
     4561 agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct
     4621 ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg
     4681 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4741 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     4801 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     4861 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     4921 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     4981 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     5041 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     5101 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     5161 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     5221 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     5281 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     5341 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     5401 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     5461 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     5521 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     5581 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     5641 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     5701 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     5761 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     5821 aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct
     5881 gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg
     5941 gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca
     6001 cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt
     6061 cggcgatata ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc
     6121 cggcgt
//
