LOCUS       sequence-333996-        4107 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express SARS-CoV-2 nsp10 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4107)
  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 4107)
  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: pCOLADuet-1_nsp10 (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..4107
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      724..1359
                     /direction=RIGHT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     1195..1214
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1377..1468
                     /gene="bla"
                     /label=AmpR promoter
     CDS             1469..2284
                     /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             1469..2284
                     /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(1542..1561)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      complement(2514..2561)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2557..2575
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2613..2657)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     protein_bind    2766..2790
                     /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)."
     promoter        complement(2791..2809)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3384..3408
                     /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)."
     promoter        complement(3409..3427)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        3473..3550
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(3551..4107,1..526)
                     /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     complement(3570..3589)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gggcccatta agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac
       61 tcgcaatcaa attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt
      121 tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa
      181 cgatcagatg gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc
      241 ggacatctcg gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt
      301 aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca
      361 actctctcag ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag
      421 aaaaaccacc ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
      481 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
      541 atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg agagccttca
      601 acccagtcag ctccttccgg tgggcgcggg gcatgactaa catgagaatt acaacttata
      661 tcgtatgggg ctgacttcag gtgctacatt tgaagagata aattgcactg aaatctagag
      721 tgatggtgtc gggaatccgt aaaggatctt cttgagatcc ttttacgatc gtcgtaatct
      781 cctgctctgt aaacgaaaaa accgcctggg gaggcggttt gatcgaaggt taagtcagtt
      841 ggggaactgc ttaacctggt aactggcttt agtggagcgc agataccaaa tactgtcctt
      901 tcagtgtagc ctctgttagg ccaccacttc aagactctcg atatctaaat ccactaattc
      961 tcagttacca atggctgctg ccagtggcgt tttgtcgtgt ctttccgggt tggactcaag
     1021 atgatagtta ccggataagg cgcagcagtc gggctgaacg gggggttctt gcacacagcc
     1081 cagcttggag cgaactgtct acacggaacg ggacgtggtg atttgggtaa agcctccacc
     1141 acaacacgga cgccgcagga cgggaacagg agagcgcaag agggagccat cagggggaaa
     1201 cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac tgatttgagc gtcagatttc
     1261 gtgatgttcg tcaggggggc ggagcctatg gaaaaacggc ttcgctccgg ccttattgtc
     1321 tctctgctaa gtatcctcct ggcatcttct aggacgtttc tgcgctagca tgcctatttg
     1381 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     1441 gcttcaataa tattgaaaaa ggaagagtat gagccatatt caacgggaaa cgtcttgctc
     1501 taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     1561 taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga
     1621 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     1681 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     1741 tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga
     1801 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     1861 gcattcgatt cctgtttgta attgtccttt taacagcgac cgcgtatttc gtctcgctca
     1921 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     1981 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     2041 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     2101 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     2161 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     2221 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     2281 ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2341 gggttccgcg cacatttccc cgaaaagtgc cacttgcgga gacccggtcg tcagcttgtc
     2401 gtcggttcag ggcagggtcg ttaaatagcc gcttatgtct attgctggtt taccggttta
     2461 ttgactaccg gaagcagtgt gaccgtgtgc ttctcaaatg cctgaggttt cagcaaaaaa
     2521 cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca
     2581 gcagcctagg ttaattaagc tgcgctagta gacgagtcca tgtgctggcg ttcaaatttc
     2641 gcagcagcgg tttctttacc agactcgagg gtaccgacgt cagcgatcgc gtggccggcc
     2701 gatatccaat tgagatctgc catatgtata tctccttctt atacttaact aatatactaa
     2761 gatggggaat tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgattat
     2821 gcggccgtgt acaatacgat tactttctgt tcgacttaag cattatgcgg ccgcaagctt
     2881 gtcgacctgc aggcgcgccg agctcgaatt cggatcctta ctgcagcatc ggttcacgca
     2941 gctgatcaca gctacaacca taacctttcc acataccaca aacggtgcaa acggtatttt
     3001 tcagggtaaa accaaccgga tcatttgcac aggtggtcgg aatctgcaca tacttacctt
     3061 tcagatcaca aaaacctttc ggattcgggt gatcaatatg acaacgacaa tacagacaac
     3121 agcttgcacc accaaagctt tcttgatcca tatttgcttc cggtgtaacg gtaattgcct
     3181 gaccggtgcc ggtatgggta cacagcattt taacacaatt ggtaatcggc tgaccaccgc
     3241 ttgccagata gtctttatag gcttttgctg catcaactgc aaatgcacaa aagctcagaa
     3301 cggtgctatt tgccggaact tcggttgcat tacctgccat ggtatatctc cttattaaag
     3361 ttaaacaaaa ttatttctac aggggaattg ttatccgctc acaattcccc tatagtgagt
     3421 cgtattaatt tcctaatgca ggagtcgcat aagggagagc gtcgagatcc cggacaccat
     3481 cgaatggcgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag
     3541 ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta
     3601 tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa
     3661 agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc
     3721 gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc
     3781 gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc
     3841 gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca
     3901 acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga
     3961 agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa
     4021 cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt
     4081 gggtcaccag caaatcgcgc tgttagc
//
