LOCUS       sequence-461580-        6129 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the fusion of the NS2B-NS3 protease from 
            Rocio virus for biochemical assay.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6129)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6129)
  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: ROCV strain SPH 34675 NS2B-NS3 protease fusion...
FEATURES             Location/Qualifiers
     source          1..6129
                     /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             1023..1040
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1093..1111)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1107..1154
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1191..1646
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1278..1297)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1488..1509
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1738..2553)
                     /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(1738..2553)
                     /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     2461..2480
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2545..2564
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2675..3263
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3164..3183
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3417..3434
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3449..3588
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3514..3533)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3674..3696
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3690..3881)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4690..5772)
                     /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     5734..5753
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5773..5850)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6056..6075
                     /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 ggacatgtgg
      301 atcgagcgtt gtgcagatgt gacgtgggag gaagaggcag agattacagg cacctccccg
      361 cgtctggatg tggaacttga cgataatggt gacttcaaaa tgatcaacga tccgggagtt
      421 ccaatgtggg ctggtggcgg aggcagcggg ggcggtgggg gcgttctttg ggacctgccg
      481 gcgcccaaac aaatgggccg ttcagatatg aaaccaggtg tatatcgtgt tatgacatct
      541 ggtgttttag gttcgtatca gtcaggggtt ggcgtgatgt acgatggagt ctttcatacc
      601 atgtggcacg ttacccaggg tgcagcactg cgcaacggtg aggggcgact gaatcctaca
      661 tggggctcgg ttcgcgatga tctgatcact tatggcggaa aatggaaact tagtgcaact
      721 tgggatggga cagaggaggt tcagcttatt gccgctgagc cgggcaaacc cgtcaagaac
      781 tttcagaccc gtccgggtgt ttttaagacg ccagccgggg aagttggagc catcaccctg
      841 gactttccga aaggtacctc gggctcgccc atcgtgaata aagctggtgc tgtcatcggg
      901 ttgtatggca atggcctcgt tctgtctcat ggagcgtacg tatcagctat aagccagggc
      961 gaacgccagg aagaagaata atgactcgag cacccgtcga caagcttgcg gccgcactcg
     1021 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     1081 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     1141 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     1201 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     1261 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     1321 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     1381 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     1441 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     1501 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     1561 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     1621 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     1681 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     1741 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     1801 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     1861 gatggcaaga tcctggtatc ggtctgctat tccgactcgt ccaacatcaa tacaacctat
     1921 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     1981 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     2041 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     2101 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     2161 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     2221 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     2281 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     2341 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     2401 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     2461 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     2521 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     2581 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     2641 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     2701 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     2761 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     2821 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     2881 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     2941 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3001 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3061 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3121 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3181 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3241 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3301 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3361 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3421 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3481 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3541 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     3601 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3661 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3721 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     3781 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     3841 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     3901 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     3961 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4021 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4081 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4141 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4201 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4261 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4321 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     4381 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     4441 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagtg gtcctcgccg
     4501 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     4561 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     4621 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     4681 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     4741 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     4801 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     4861 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     4921 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     4981 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     5041 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     5101 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     5161 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     5221 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     5281 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     5341 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     5401 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     5461 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5521 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     5581 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     5641 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     5701 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     5761 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     5821 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     5881 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     5941 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     6001 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     6061 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     6121 gtccggcgt
//
