LOCUS       sequence-333225-        6139 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 6139)
  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 6139)
  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: pET11a_3xFlag-nsp10 (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..6139
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     549..568
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(588..665)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     871..890
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     974..993
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        974..992
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    993..1017
                     /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             1032..1054
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1065..1130
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(1613..1631)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1627..1674
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1849..1854,1855..1871,1872..1877))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1904..1922)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1990..2094
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2095..2163,2164..2955)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2313..2332)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3126..3714
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3615..3634
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3868..3885
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3900..4039
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3965..3984)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4125..4147
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4141..4332)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5644..6139,1..587))
                     /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 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
       61 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      121 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      181 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      241 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      301 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      361 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      421 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      481 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      541 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
      601 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
      661 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
      721 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
      781 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
      841 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
      901 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
      961 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1021 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatggactat aaagatcacg
     1081 acggcgatta taaagaccat gacatcgatt acaaagatga cgatgataaa gttcgtctgc
     1141 aggcaggtaa tgcaaccgaa gttccggcaa atagcaccgt tctgagcttt tgtgcatttg
     1201 cagttgatgc agcaaaagcc tataaagact atctggcaag cggtggtcag ccgattacca
     1261 attgtgttaa aatgctgtgt acccataccg gcaccggtca ggcaattacc gttacaccgg
     1321 aagcaaatat ggatcaagaa agctttggtg gtgcaagctg ttgtctgtat tgtcgttgtc
     1381 atattgatca cccgaatccg aaaggttttt gtgatctgaa aggtaagtat gtgcagattc
     1441 cgaccacctg tgcaaatgat ccggttggtt ttaccctgaa aaataccgtt tgcaccgttt
     1501 gtggtatgtg gaaaggttat ggttgtagct gtgatcagct gcgtgaaccg atgctgcagt
     1561 aaggatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1621 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1681 ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata accaagccta
     1741 tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca
     1801 tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat
     1861 cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta
     1921 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     1981 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     2041 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     2101 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     2161 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     2221 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     2281 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     2341 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2401 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2461 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2521 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2581 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     2641 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2701 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2761 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     2821 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2881 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2941 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     3001 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     3061 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     3121 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     3181 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     3241 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3301 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3361 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3421 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3481 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3541 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3601 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3661 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3721 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     3781 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     3841 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     3901 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     3961 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     4021 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     4081 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     4141 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     4201 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     4261 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     4321 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     4381 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     4441 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     4501 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     4561 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     4621 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     4681 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     4741 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     4801 ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat
     4861 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc
     4921 tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag
     4981 gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg
     5041 cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg
     5101 atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt
     5161 ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg
     5221 ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc
     5281 agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg
     5341 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     5401 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     5461 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     5521 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     5581 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     5641 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5701 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     5761 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     5821 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     5881 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     5941 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     6001 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     6061 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     6121 atgccagcca gccagacgc
//
