LOCUS       sequence-464103-        6076 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the A71 2A protease with ITTL at the 
            N-terminal and a C110A mutation used for crystallization in P 6 1 
            (PDB 9FGO).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6076)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6076)
  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: EV-A71 isolate 4643-TW98 2A precursor protease...
FEATURES             Location/Qualifiers
     source          1..6076
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     54..73
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        54..72
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    73..97
                     /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             112..134
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             154..171
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             949..993
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             994..1011
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1064..1082)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1078..1125
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1162..1617
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1249..1268)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1459..1480
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1709..2524)
                     /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(1709..2524)
                     /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     2432..2451
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2516..2535
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2646..3234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3135..3154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3388..3405
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3420..3559
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3485..3504)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3645..3667
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3661..3852)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4661..5743)
                     /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     5705..5724
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5744..5821)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6027..6046
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
       61 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
      121 ctttaagaag gagatataca tatgtgcagc agccatcatc atcatcatca cggcagcggc
      181 agcggctctg accaggaggc aaaaccttca actgaggact tgggggataa gaaggaaggt
      241 gaatatatta aactcaaagt cattggacag gatagcagtg agattcactt caaagtgaaa
      301 atgacaacac atctcaagaa actcaaagaa tcatactgtc aaagacaggg tgttccaatg
      361 aattcactca ggtttctctt tgagggtcag agaattgctg ataatcatac tccaaaagaa
      421 ctgggaatgg aggaagaaga tgtgattgaa gtttaccagg agcaaacggg aggtatcacc
      481 accctcggga aatttggaca gcagtccgga gctatctacg tgggcaactt tagagtggtt
      541 aaccgccatc ttgctactca taatgactgg gcaaaccttg tttgggaaga cagctcccgc
      601 gacttgctcg tatcatctac cactgctcaa ggttgtgaca cgattgctcg ttgcaattgc
      661 cagacaggag tgtattattg taactcaatg agaaaacact atccggtcag tttctcgaaa
      721 cccagtttga tcttcgtgga ggccagcgag tattatccag ctagatacca gtcacatctc
      781 atgcttgcag tgggtcattc ggaaccaggg gatgccggtg gcattcttag atgccaacat
      841 ggcgtcgtag ggatagtttc caccggggga aacggcctgg tggggttcgc cgatgtgagg
      901 gatcttctgt ggttggatga tgaagccatg gagcagtaaa gcggtggcgg tctgaacgac
      961 atcttcgagg ctcagaaaat cgaatggcac gaacaccacc accaccacca ctgagatccg
     1021 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1081 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1141 atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1201 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1261 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     1321 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     1381 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg
     1441 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct
     1501 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg
     1561 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg
     1621 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc
     1681 aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact
     1741 gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
     1801 aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga
     1861 ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat
     1921 caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca
     1981 tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat
     2041 caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
     2101 taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat
     2161 caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg
     2221 ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg
     2281 gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg
     2341 caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
     2401 gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat
     2461 cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc
     2521 tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa
     2581 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2641 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2701 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2761 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     2821 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2881 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2941 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3001 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3061 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3121 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3181 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3241 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3301 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3361 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3421 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     3481 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     3541 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     3601 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3661 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     3721 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     3781 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     3841 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     3901 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     3961 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     4021 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4081 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4141 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4201 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4261 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4321 ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt
     4381 gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat
     4441 cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg
     4501 tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg
     4561 cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg
     4621 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     4681 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4741 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     4801 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     4861 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     4921 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     4981 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     5041 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     5101 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     5161 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     5221 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     5281 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     5341 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     5401 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     5461 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     5521 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     5581 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     5641 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     5701 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     5761 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt
     5821 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
     5881 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
     5941 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
     6001 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
     6061 cctgtggcgc cggtga
//
