LOCUS       sequence-333990-        6304 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express nsp10-nsp14 fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6304)
  AUTHORS   Canal B, McClure AW, Curran JF, Wu M, Ulferts R, Weissmann F, Zeng 
            J, Bertolin AP, Milligan JC, Basu S, Drury LS, Deegan TD, Fujisawa 
            R, Roberts EL, Basier C, Labib K, Beale R, Howell M, Diffley JFX
  TITLE     Identifying SARS-CoV-2 antiviral compounds by screening for small 
            molecule inhibitors of nsp14/nsp10 exoribonuclease.
  JOURNAL   Biochem J. 2021 Jul 16;478(13):2445-2464. doi: 10.1042/BCJ20210198.
  PUBMED    34198326
REFERENCE   2  (bases 1 to 6304)
  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: "2445-2464"
COMMENT     Sequence Label: pK27Sumo_His-SUMO-nsp10-nsp14 fusion (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..6304
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1004..1023
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1043..1120)
                     /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(1204..1226)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1312..1452
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1367..1386
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1467..1484)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1638..2226)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1718..1737)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2317..2335
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2359..2373,2374..2379,2380..2396,2397..2403)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    2379..2395
                     /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    2411..2427
                     /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             2446..2457
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2542..2832
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     terminator      4898..4992
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(5040..5059)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5051..5866
                     /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             5051..5866
                     /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(5124..5143)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      6085..6171
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(join(6264..6304,1..1042))
                     /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 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggg gccagggtgg
       61 tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag
      121 agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg
      181 tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga
      241 tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct
      301 gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt
      361 gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat
      421 tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg
      481 ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc
      541 gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa
      601 gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca
      661 gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt
      721 tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat
      781 cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg
      841 tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa
      901 tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc
      961 tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat
     1021 cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc
     1081 atgccatacc gcgaaaggtt ttgcaccatt cgatggtgtc ggaatttcgg gcagcgttgg
     1141 gtcctggcca cgggtgcgca tgatctagag ctgcctcgcg cgtttcggtg atgacggtga
     1201 aaacctctga cacatgcagc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     1261 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     1321 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     1381 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     1441 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1501 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1561 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1621 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1681 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1741 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1801 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1861 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     1921 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     1981 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2041 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     2101 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2161 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2221 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2281 cgttaaggga ttttggtcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     2341 cgtcttcacc tcgagaaatc ataaaaaatt tatttgcttt gtgagcggat aacaattata
     2401 atagattcaa ttgtgagcgg ataacaattt cacacagaat tcattaaaga ggagaaatta
     2461 accatgagca agcatcacca tcattcaggc catcaccata ccggacacca ccatcattca
     2521 ggcagtcatc accataccgg atcggactca gaagtcaatc aagaagctaa gccagaggtc
     2581 aagccagaag tcaagcctga gactcacatc aatttaaagg tgtccgatgg atcttcagag
     2641 atcttcttca agatcaaaaa gaccactcct ttaagaaggc tgatggaagc gttcgctaaa
     2701 agacagggta aggaaatgga ctccttaaga ttcttgtacg acggtattag aattcaagct
     2761 gatcagaccc ctgaagattt ggacatggag gataacgata ttattgaggc tcacagagaa
     2821 cagattggtg gcgcaggtaa tgcaaccgaa gttccggcaa atagcaccgt tctgagcttt
     2881 tgtgcatttg cagttgatgc agcaaaagcc tataaagact atctggcaag cggtggtcag
     2941 ccgattacca attgtgttaa aatgctgtgt acccataccg gcaccggtca ggcaattacc
     3001 gttacaccgg aagcaaatat ggatcaagaa agctttggtg gtgcaagctg ttgtctgtat
     3061 tgtcgttgtc atattgatca cccgaatccg aaaggttttt gtgatctgaa aggtaagtat
     3121 gtgcagattc cgaccacctg tgcaaatgat ccggttggtt ttaccctgaa aaataccgtt
     3181 tgcaccgttt gtggtatgtg gaaaggttat ggttgtagct gtgatcagct gcgtgaaccg
     3241 atgctgcagg gtggtagcgg tggtagtgca gaaaatgtta ccggtctgtt taaagattgc
     3301 agcaaagtta ttacaggtct gcatccgaca caggcaccga cacatctgag cgttgatacc
     3361 aaattcaaaa ccgaaggtct gtgtgttgat attccgggta ttccgaaaga tatgacctat
     3421 cgtcgtctga ttagcatgat gggcttcaaa atgaattatc aggtgaatgg ctacccgaac
     3481 atgtttatta cccgtgaaga agcaattcgt cacgttcgtg catggattgg ttttgatgtt
     3541 gaaggttgtc atgcaacacg tgaagcagtt ggcaccaatc tgccgctgca gctgggtttt
     3601 agcaccggtg ttaatctggt tgcagttccg accggttatg ttgatacccc gaataacacc
     3661 gattttagcc gtgttagcgc aaaaccgcct ccgggtgatc agtttaaaca tctgattccg
     3721 ctgatgtata aaggtctgcc gtggaatgtt gtgcgtatca aaattgttca gatgctgagc
     3781 gataccctga aaaatctgag cgatcgtgtt gtttttgttc tgtgggcaca tggttttgaa
     3841 ctgaccagca tgaaatactt cgtgaaaatt ggtccggaac gtacctgttg tctgtgtgat
     3901 cgtcgtgcaa cctgttttag taccgcaagc gatacctatg catgttggca tcatagcatt
     3961 ggcttcgatt atgtgtataa cccgtttatg attgatgttc agcagtgggg ttttaccggc
     4021 aatctgcaga gcaatcatga tctgtattgt caggttcatg gtaatgcaca tgttgcaagc
     4081 tgtgatgcaa ttatgacccg ttgtctggca gttcatgaat gctttgttaa acgtgttgat
     4141 tggaccatcg aatatccgat tattggtgat gagctgaaaa ttaacgcagc ctgtcgtaaa
     4201 gttcagcaca tggtggttaa agcagcactg ctggcagata aatttccggt tctgcatgat
     4261 attggtaacc cgaaagcaat taaatgtgtt ccgcaggcag atgtggaatg gaaattctat
     4321 gatgcacagc cgtgtagcga taaagcctat aaaatcgaag aactgttcta tagctatgcg
     4381 acccatagcg acaaatttac cgatggtgtt tgtctgtttt ggaattgcaa tgttgatcgc
     4441 tatccggcaa atagcattgt ttgccgtttt gatacccgtg ttctgagcaa tctgaatctg
     4501 cctggttgtg atggtggtag cctgtatgtt aacaaacatg catttcatac ccctgccttt
     4561 gataaaagcg catttgtgaa tctgaaacag ctgccgtttt tctattatag cgatagcccg
     4621 tgtgaaagcc atggtaaaca ggtggttagc gatattgatt atgttccgct gaaaagcgca
     4681 acctgcatta cccgttgtaa tcttggtggt gcagtttgtc gtcatcatgc aaatgaatat
     4741 cgtctgtatc tggatgccta caacatgatg attagcgcag gttttagcct gtgggtgtat
     4801 aaacagtttg atacctataa tctgtggaac acctttacac gtctgcagta ataagcggcc
     4861 gcacatcacc atcactaagc ttaattagct gagcttggac tcctgttgat agatccagta
     4921 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
     4981 attggtgaga atccaagcta gccatgaaaa taaactgtct gcttacataa acagtaatac
     5041 aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc gattaaattc
     5101 caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg ggcaatcagg
     5161 tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg
     5221 caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact ggctgacgga
     5281 atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg cttggttact
     5341 cacgactgcg atccccggca aaacagcatt ccaggtatta gaagaatatc ctgattcagg
     5401 tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg
     5461 taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa
     5521 taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc ctgttgaaca
     5581 agtctggaaa gaaatgcaca aacttttgcc attctcaccg gattcagtcg tcactcatgg
     5641 tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt gtattgatgt
     5701 tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga actgcctcgg
     5761 tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg ataatcctga
     5821 tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat taattcatgg
     5881 gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg
     5941 ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg
     6001 agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa acgcctgggg
     6061 taatgactct ctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     6121 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccctct
     6181 agattacgtg cagtcgatga taagctgtca aacatgagaa ttgtgcctaa tgagtgagct
     6241 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6301 agct
//
