LOCUS       sequence-461428-        6150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the A71 3C protease (isoform B) used in 
            crystallisation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6150)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6150)
  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: Enterovirus A71 (EV-A71) isolate TW-2553-1998 ...
FEATURES             Location/Qualifiers
     source          1..6150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..40
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    40..64
                     /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)."
     CDS             121..138
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1035..1052
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1105..1123)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1119..1166
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1203..1658
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1290..1309)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1500..1521
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1750..2565)
                     /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(1750..2565)
                     /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     2473..2492
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2557..2576
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2687..3275
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3176..3195
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3429..3446
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3461..3600
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3526..3545)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3686..3708
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3702..3893)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4702..5784)
                     /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     5746..5765
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5785..5862)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6068..6087
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
       61 ttcccctcta gaaataattt tgtttaacct taagaaggag atatacacat gggttcgagt
      121 catcaccacc accaccacat ggcgtccatg tccgatagcg aagttaacca ggaagccaaa
      181 cctgaggtga aacctgaggt caagcctgag actcatataa acttgaaagt ttccgacggc
      241 agttccgaaa tcttcttcaa gattaagaag actacaccgt tacggcggtt gatggaagca
      301 ttcgccaagc gacaaggcaa agagatggac tctcttcgtt ttctctacga cggcatacgg
      361 atacaagcag atcaaacacc cgaggacctt gacatggaag acaacgacat aatcgaggcg
      421 cacagagaac agattggtgg cggtccctct ctggacttcg cactcagtct gctccgtcgt
      481 aatatccgtc aagtgcaaac cgatcaaggc catttcacta tgctgggtgt tcgagatcgc
      541 ctggctgtat tgccccgtca cagccagccg ggtaaaacta tctgggtcga gcacaagtta
      601 gtgaagatcg tcgatgccgt tgagctcgtg gatgaacaag gggtgaacct cgagttgaca
      661 ttagttacgt tagatacaaa cgaaaagttc cgggacatta ctcggttcat acctgaaacc
      721 ataagccctg cttcggacgc gacccttgtt atcaacacag agcatatgcc gagtatgttt
      781 gtaccagtag gcgatgtggt acaatacggc tttctcaatc tctccgggaa gcctacacat
      841 cggaccatga tgtataactt cccaacgaag gcaggacaat gtggcggggt tgttacagca
      901 gttggaaaag ttattgggat tcatataggt ggaaacggcc gccagggatt ttgtgctgct
      961 ttgaagagag ggtatttctg tagtgagcaa taatgactcg agcacccgtc gacaagcttg
     1021 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     1081 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1141 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     1201 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     1261 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     1321 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     1381 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     1441 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     1501 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     1561 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     1621 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     1681 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     1741 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     1801 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     1861 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     1921 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     1981 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     2041 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     2101 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     2161 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     2221 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     2281 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     2341 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     2401 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     2461 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     2521 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     2581 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     2641 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     2701 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     2761 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     2821 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     2881 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     2941 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3001 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3061 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3121 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3181 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3241 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3301 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3361 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     3421 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     3481 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     3541 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     3601 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     3661 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     3721 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     3781 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     3841 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     3901 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     3961 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     4021 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     4081 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     4141 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4201 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     4261 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     4321 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     4381 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     4441 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     4501 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     4561 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     4621 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     4681 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4741 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     4801 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     4861 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     4921 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     4981 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5041 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5101 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5161 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     5221 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     5281 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     5341 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     5401 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     5461 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     5521 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     5581 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     5641 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     5701 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     5761 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     5821 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     5881 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     5941 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6001 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6061 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6121 cggccacgat gcgtccggcg tagaggatcg
//
