LOCUS       sequence-461401-        6015 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the Coxsackievirus A16 (G-10) 2A protease 
            this construct can be used for biochemical assay and to obtain 
            crystals in the space group C1 2 1(PDB 8POA).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6015)
  TITLE     ASAP Discovery Plasmids 2024
REFERENCE   2  (bases 1 to 6015)
  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: Human Coxsackievirus A16 strain G10 2A protease
FEATURES             Location/Qualifiers
     source          1..6015
                     /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)."
     RBS             78..100
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             111..128
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             899..916
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(969..987)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      983..1030
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1067..1522
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1154..1173)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1364..1385
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1614..2429)
                     /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(1614..2429)
                     /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     2337..2356
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2421..2440
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2551..3139
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3040..3059
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3293..3310
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3325..3464
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3390..3409)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3550..3572
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3566..3757)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4566..5648)
                     /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     5610..5629
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5649..5726)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5932..5951
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
       61 tcccctctag aaataatttt gtttaacttt aagaaggaga tatacatatg caccaccatc
      121 atcaccatgg ctcaggagat caggaagcga aaccttcgac cgaggacctg ggcgacaaga
      181 aagaagggga gtatattaag ttaaaggtga tcggccagga ctcaagcgag atccacttca
      241 aagtcaaaat gaccacgcac ctcaagaagt tgaaggagag ttactgtcaa cggcaagggg
      301 ttcctatgaa ctcgcttcgc ttcctgttcg agggccagcg aattgcggac aaccacacac
      361 ctaaggaact tggtatggaa gaggaagacg tcattgaggt gtaccaggag cagacaggcg
      421 gctcgggtgc catatatgtg ggtaactatc gtgtggttaa tcgccacctc gctactcaca
      481 atgattgggc caacttagtg tgggaggaca gctctcgcga tctccttgta tcatcgacca
      541 ctgcgcaggg ttgcgacacc attgcgcgct gtgattgcca gacaggcgtg tattattgct
      601 cgtctcgtcg taaacattat ccggtttctt tttctaagcc gtcactgatc tttgttgaag
      661 cgtcagagta ctatccagcc cgttatcagt cccatctgat gctggctgtg ggtcacagcg
      721 aacccgggga ttgtggcggt atactgcgct gtcagcacgg tgtagttggc attgtcagta
      781 caggaggtaa tgggttagtc ggatttgccg atgtgcggga tttactgtgg ttggacgagg
      841 aggctatgga acagtaatga ctcgagcacc cgtcgacaag cttgcggccg cactcgagca
      901 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
      961 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1021 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     1081 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     1141 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     1201 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     1261 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     1321 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     1381 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     1441 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     1501 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1561 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     1621 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     1681 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     1741 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     1801 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     1861 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     1921 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     1981 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     2041 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     2101 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     2161 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     2221 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     2281 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     2341 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     2401 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     2461 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     2521 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     2581 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     2641 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     2701 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     2761 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     2821 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     2881 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     2941 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     3001 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     3061 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     3121 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     3181 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     3241 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     3301 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     3361 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     3421 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     3481 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     3541 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     3601 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     3661 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     3721 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     3781 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     3841 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     3901 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     3961 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4021 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4081 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4141 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     4201 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     4261 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     4321 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagtggtcc tcgccgaaaa
     4381 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     4441 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     4501 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     4561 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     4621 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     4681 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     4741 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     4801 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     4861 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     4921 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     4981 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     5041 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     5101 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     5161 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     5221 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     5281 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     5341 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     5401 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     5461 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     5521 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     5581 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     5641 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     5701 aaggttttgc gccattcgat ggtgtccggg atctcgacgc tctcccttat gcgactcctg
     5761 cattaggaag cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg
     5821 tgcatgcaag gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac
     5881 gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc
     5941 ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc
     6001 ggcgtagagg atcga
//
