LOCUS       sequence-461604-        6659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial co-expression of the NS2B and NS3 proteases from Zika 
            virus for crystallization assays in P43 2 2 (PDB:8PN6).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6659)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6659)
  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: ZIKV isolate VE_Ganxian2016 NS2B-NS3 protease
            ...
FEATURES             Location/Qualifiers
     source          1..6659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     52..71
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        52..70
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    71..95
                     /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             110..132
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             143..160
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             167..820
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             836..856
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1575..1592
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1645..1663)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1659..1706
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1743..2198
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1830..1849)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2040..2061
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2290..3105)
                     /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(2290..3105)
                     /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     3013..3032
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3097..3116
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3227..3815
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3716..3735
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3969..3986
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4001..4140
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4066..4085)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4226..4248
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4242..4433)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5242..6324)
                     /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     6286..6305
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6325..6402)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6608..6627
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
       61 tcactatagg ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact
      121 ttaagaagga gatatacata tgcaccatca ccaccatcac tcatcgatgt ctcccatttt
      181 gggttattgg aagataaagg ggttagtaca accgaccaga ttactgttag agtaccttga
      241 agagaagtat gaagagcatt tatatgagcg cgacgaaggc gataagtggc gcaacaagaa
      301 atttgagtta ggcctcgagt ttccgaacct tccttattat atcgacgggg acgtaaagtt
      361 gacgcaatct atggcaataa ttcggtatat agcagacaag cataacatgt tgggtggctg
      421 ccccaaagag cgtgccgaga tctccatgtt agaaggagcc gtcctggaca ttcgctatgg
      481 tgtgtcgcgc attgcctact caaaagattt cgagacttta aaagttgact ttctgagcaa
      541 gctgccggag atgcttaaga tgttcgaaga tcggctgtgt cacaagacgt acctcaacgg
      601 tgaccacgtt acccacccag actttatgct ttacgatgcg ctggacgttg tgctttacat
      661 ggacccgatg tgtctggacg cgtttccgaa gttggtatgt ttcaagaagc ggattgaggc
      721 tattccccaa atagacaaat accttaagag tagtaagtac atcgcgtggc cacttcaagg
      781 ttggcaagct accttcggcg gcggcgacca tcccccaaag tcgggcggcg gcagcgagaa
      841 cctgtacttc caatccatgg gaaagtcagt tgatatgtac atagaacggg ctggagatat
      901 tacctgggaa aaagacgcag aagtgacggg caacagcccg cggttggatg ttgctttaga
      961 cgaatctgga gattttagct tagttgaaga ataatgaaaa taattttatt tataaggaga
     1021 tataccatga aggaggtcaa aaaaggtgag actactgatg gtgtttaccg agtgatgacc
     1081 cgccgtcttc tgggttccac gcaagtaggc gtgggggtta tgcaggaggg tgtttttcat
     1141 actatgtggc atgtaaccaa aggtgctgca cttcgcagtg gagaaggacg tcttgaccca
     1201 tactggggcg atgtaaaaca ggatttggtt agttattgcg gaccgtggaa attggatgcc
     1261 gcgtgggacg gcctttccga agtccagttg ctggcagttc caccaggaga acgcgctaag
     1321 aacatacaaa ctctgcctgg tatatttaaa accaaagatg gtgacattgg cgcggtggct
     1381 ttagactacc ccgccggcac gagcgggtcc ccgatcttag ataagtgcgg tcgtgtaatc
     1441 ggcctgtacg gcaatggcgt tgtaattaag aacggcagtt atgtgtctgc gataactcag
     1501 ggtaagcgcg aagaggaaac gcctgttgaa taatgactcg agcacccgtc gacaagcttg
     1561 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     1621 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1681 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     1741 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1801 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1861 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1921 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1981 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2041 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2101 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2161 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     2221 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2281 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     2341 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     2401 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     2461 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     2521 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     2581 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     2641 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     2701 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     2761 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     2821 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     2881 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     2941 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     3001 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     3061 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     3121 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     3181 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3241 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3301 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3361 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3421 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3481 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3541 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3601 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3661 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3721 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3781 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3841 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3901 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3961 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4021 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     4081 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     4141 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4201 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4261 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     4321 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     4381 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     4441 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     4501 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     4561 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     4621 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     4681 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4741 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     4801 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4861 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4921 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4981 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     5041 tggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     5101 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     5161 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     5221 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5281 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     5341 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5401 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     5461 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5521 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5581 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5641 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5701 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5761 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5821 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5881 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5941 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     6001 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     6061 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     6121 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     6181 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     6241 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     6301 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     6361 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     6421 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6481 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6541 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6601 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatg
//
