LOCUS       sequence-333984-        5617 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express nsp10-nsp16 fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5617)
  AUTHORS   Basu S, Mak T, Ulferts R, Wu M, Deegan T, Fujisawa R, Tan KW, Lim 
            CT, Basier C, Canal B, Curran JF, Drury LS, McClure AW, Roberts EL, 
            Weissmann F, Zeisner TU, Beale R, Cowling VH, Howell M, Labib K, 
            Diffley JFX
  TITLE     Identifying SARS-CoV-2 antiviral compounds by screening for small 
            molecule inhibitors of Nsp14 RNA cap methyltransferase.
  JOURNAL   Biochem J. 2021 Jul 16;478(13):2481-2497. doi: 10.1042/BCJ20210219.
  PUBMED    34198328
REFERENCE   2  (bases 1 to 5617)
  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"; doi: "10"; journalName: 
            "Biochem J"; date: "2021-07-16- 16"; volume: "478"; issue: "13"; 
            pages: "2481-2497"
COMMENT     Sequence Label: pK27Sumo_His-SUMO-nsp10-nsp16 fusion (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..5617
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     965..984
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1004..1081)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(1165..1187)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1273..1413
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1328..1347
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1428..1445)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1599..2187)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1679..1698)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2278..2296
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2320..2334,2335..2340,2341..2357,2358..2364)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    2340..2356
                     /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)."
     protein_bind    2372..2388
                     /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             2407..2418
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2503..2793
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     terminator      4172..4266
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(4314..4333)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4325..5140
                     /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             4325..5140
                     /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     complement(4398..4417)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      5359..5445
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(5538..5617,1..1003))
                     /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"
ORIGIN
        1 gcgtattggg ggccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
       61 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
      121 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
      181 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
      241 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
      301 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
      361 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
      421 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
      481 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
      541 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
      601 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
      661 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
      721 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
      781 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
      841 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
      901 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
      961 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     1021 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcaccat tcgatggtgt
     1081 cggaatttcg ggcagcgttg ggtcctggcc acgggtgcgc atgatctaga gctgcctcgc
     1141 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggcat ccgcttacag
     1201 acaagctgtg accgtctccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     1261 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     1321 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     1381 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     1441 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     1501 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     1561 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1621 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1681 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1741 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1801 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1861 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1921 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1981 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     2041 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     2101 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     2161 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2221 gctcagtgga acgaaaactc acgttaaggg attttggtca tgacattaac ctataaaaat
     2281 aggcgtatca cgaggccctt tcgtcttcac ctcgagaaat cataaaaaat ttatttgctt
     2341 tgtgagcgga taacaattat aatagattca attgtgagcg gataacaatt tcacacagaa
     2401 ttcattaaag aggagaaatt aaccatgagc aagcatcacc atcattcagg ccatcaccat
     2461 accggacacc accatcattc aggcagtcat caccataccg gatcggactc agaagtcaat
     2521 caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat caatttaaag
     2581 gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc tttaagaagg
     2641 ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag attcttgtac
     2701 gacggtatta gaattcaagc tgatcagacc cctgaagatt tggacatgga ggataacgat
     2761 attattgagg ctcacagaga acagattggt ggcgcaggta atgcaaccga agttccggca
     2821 aatagcaccg ttctgagctt ttgtgcattt gcagttgatg cagcaaaagc ctataaagac
     2881 tatctggcaa gcggtggtca gccgattacc aattgtgtta aaatgctgtg tacccatacc
     2941 ggcaccggtc aggcaattac cgttacaccg gaagcaaata tggatcaaga aagctttggt
     3001 ggtgcaagct gttgtctgta ttgtcgttgt catattgatc acccgaatcc gaaaggtttt
     3061 tgtgatctga aaggtaagta tgtgcagatt ccgaccacct gtgcaaatga tccggttggt
     3121 tttaccctga aaaataccgt ttgcaccgtt tgtggtatgt ggaaaggtta tggttgtagc
     3181 tgtgatcagc tgcgtgaacc gatgctgcag ggtagcggta gtggtagcag cagccaggca
     3241 tggcagcctg gtgttgcaat gccgaatctg tataaaatgc agcgtatgct gctggaaaaa
     3301 tgcgatctgc agaattatgg tgatagcgca accctgccga aaggtattat gatgaatgtt
     3361 gcaaaatata cccagctgtg ccagtatctg aataccctga cactggcagt tccgtataac
     3421 atgcgtgtta ttcattttgg tgcaggtagc gataaaggtg ttgcaccggg tacagcagtt
     3481 ctgcgtcagt ggctgccgac cggtacactg ctggttgata gcgatctgaa tgattttgtt
     3541 agtgatgcag atagcaccct gattggtgat tgtgcaaccg ttcataccgc aaataaatgg
     3601 gatctgatta tcagcgatat gtacgacccg aaaaccaaaa atgtgaccaa agagaacgat
     3661 agcaaagaag gctttttcac ctatatctgc ggtttcattc agcagaaact ggcattaggt
     3721 ggtagcgttg caatcaaaat taccgaacat agctggaatg cggatctgta caaactgatg
     3781 ggtcattttg catggtggac cgcatttgtt accaatgtta atgcaagcag cagcgaagca
     3841 tttctgattg gttgtaatta tctgggtaaa ccgcgtgagc agattgatgg ttatgttatg
     3901 catgccaact atatcttttg gcgcaatacc aatccgattc agctgagcag ctatagcctg
     3961 tttgatatga gcaaatttcc gctgaaactg cgtggcaccg cagttatgag cctgaaagaa
     4021 ggtcagatta acgatatgat tctgagcctg ctgagcaaag gtcgtctgat tattcgtgaa
     4081 aataatcgcg ttgttattag ctcagatgtg ctggtgaata attaataagc ggccgcacat
     4141 caccatcact aagcttaatt agctgagctt ggactcctgt tgatagatcc agtaatgacc
     4201 tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt
     4261 gagaatccaa gctagccatg aaaataaact gtctgcttac ataaacagta atacaagggg
     4321 tgttatgagc catattcaac gggaaacgtc ttgctctagg ccgcgattaa attccaacat
     4381 ggatgctgat ttatatgggt ataaatgggc tcgcgataat gtcgggcaat caggtgcgac
     4441 aatctatcga ttgtatggga agcccgatgc gccagagttg tttctgaaac atggcaaagg
     4501 tagcgttgcc aatgatgtta cagatgagat ggtcagacta aactggctga cggaatttat
     4561 gcctcttccg accatcaagc attttatccg tactcctgat gatgcttggt tactcacgac
     4621 tgcgatcccc ggcaaaacag cattccaggt attagaagaa tatcctgatt caggtgaaaa
     4681 tattgttgat gcgctggcag tgttcctgcg ccggttgcat tcgattcctg tttgtaattg
     4741 tccttttaac agcgatcgcg tatttcgtct cgctcaggcg caatcacgaa tgaataacgg
     4801 tttggttgat gcgagtgatt ttgatgacga gcgtaatggc tggcctgttg aacaagtctg
     4861 gaaagaaatg cacaaacttt tgccattctc accggattca gtcgtcactc atggtgattt
     4921 ctcacttgat aaccttattt ttgacgaggg gaaattaata ggttgtattg atgttggacg
     4981 agtcggaatc gcagaccgat accaggatct tgccatccta tggaactgcc tcggtgagtt
     5041 ttctccttca ttacagaaac ggctttttca aaaatatggt attgataatc ctgatatgaa
     5101 taaattgcag tttcatttga tgctcgatga gtttttctaa gaattaattc atgggcaaat
     5161 attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt
     5221 gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc
     5281 agggcggggc gtaatttttt taaggcagtt attggtgccc ttaaacgcct ggggtaatga
     5341 ctctctagct tgaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt
     5401 tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc ctctagatta
     5461 cgtgcagtcg atgataagct gtcaaacatg agaattgtgc ctaatgagtg agctaactta
     5521 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5581 attaatgaat cggccaacgc gcggggagag gcggttt
//
