LOCUS       sequence-461410-        5766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the A71 2A protease for assays that require 
            immobilization using biotin-streptavidin interaction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5766)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 5766)
  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 4643-TW98 2A
            ...
FEATURES             Location/Qualifiers
     source          1..5766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    39..63
                     /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             543..560
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             561..605
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             650..667
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(720..738)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      734..781
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      818..1273
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(905..924)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1115..1136
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1365..2180)
                     /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(1365..2180)
                     /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     2088..2107
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2172..2191
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2302..2890
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2791..2810
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3044..3061
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3076..3215
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3141..3160)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3301..3323
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3317..3508)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4317..5399)
                     /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     5361..5380
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5400..5477)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5683..5702
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
       61 tcccctctag aaataatttt gtttaacctt aagaaggaga tatacacatg tcgggtgcca
      121 tatatgtggg taactatcgt gtggttaatc gccacctcgc tactcacaat gattgggcca
      181 acttagtgtg ggaggacagc tctcgcgatc tccttgtatc atcgaccact gcgcagggtt
      241 gcgacaccat tgcgcgctgt gattgccaga caggcgtgta ttattgctcg tctcgtcgta
      301 aacattatcc ggtttctttt tctaagccgt cactgatctt tgttgaagcg tcagagtact
      361 atccagcccg ttatcagtcc catctgatgc tggctgtggg tcacagcgaa cccggggatt
      421 gtggcggtat actgcgctgt cagcacggtg tagttggcat tgtcagtaca ggaggtaatg
      481 ggttagtcgg atttgccgat gtgcgggatt tactgtggtt ggacgaggag gctatggaac
      541 agcatcatca ccatcaccac ggcctgaacg atatttttga agcgcagaaa attgaatggc
      601 atgaataatg actcgagcac ccgtcgacaa gcttgcggcc gcactcgagc accaccacca
      661 ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac
      721 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt
      781 gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta
      841 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
      901 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
      961 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     1021 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     1081 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     1141 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     1201 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     1261 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     1321 ttttctaaat acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg
     1381 agcatcaaat gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa
     1441 agccgtttct gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc
     1501 tggtatcggt ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg
     1561 tcaaaaataa ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat
     1621 ggcaaaagtt tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca
     1681 tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga
     1741 aatacgcgat cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg
     1801 aacactgcca gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg
     1861 aatgctgttt tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata
     1921 aaatgcttga tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca
     1981 tctgtaacat cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg
     2041 ggcttcccat acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat
     2101 ttatacccat ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt
     2161 tcccgttgaa tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt
     2221 attgttcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     2281 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     2341 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     2401 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
     2461 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     2521 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     2581 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     2641 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     2701 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     2761 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     2821 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     2881 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     2941 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     3001 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     3061 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3121 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
     3181 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
     3241 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     3301 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
     3361 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
     3421 ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
     3481 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
     3541 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
     3601 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
     3661 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
     3721 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
     3781 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
     3841 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
     3901 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
     3961 tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4021 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4081 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4141 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4201 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     4261 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     4321 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     4381 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     4441 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     4501 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     4561 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     4621 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     4681 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     4741 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     4801 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     4861 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     4921 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     4981 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5041 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5101 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5161 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     5221 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     5281 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     5341 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     5401 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     5461 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     5521 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     5581 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     5641 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     5701 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     5761 gatcga
//
