LOCUS       sequence-333230-        7339 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express SARS-CoV-2 nsp14 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7339)
  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 7339)
  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-nsp14 (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..7339
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     970..989
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1009..1086)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1292..1311
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1395..1414
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1395..1413
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1414..1438
                     /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             1453..1475
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1486..1551
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             3151..3180
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="ASMTGGQQMG"
     primer_bind     complement(3234..3252)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3248..3295
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3470..3475,3476..3492,3493..3498))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3525..3543)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3611..3715
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3716..3784,3785..4576)
                     /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(3934..3953)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4747..5335
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5236..5255
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5489..5506
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5521..5660
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5586..5605)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5746..5768
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5762..5953)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7265..7339,1..1008))
                     /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 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
       61 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
      121 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
      181 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
      241 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
      301 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
      361 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
      421 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
      481 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
      541 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
      601 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
      661 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
      721 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
      781 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
      841 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
      901 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
      961 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     1021 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     1081 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     1141 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     1201 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     1261 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     1321 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     1381 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     1441 tctagaaata attttgttta actttaagaa ggagatatac atatggatta caaagatcac
     1501 gatggcgact ataaagacca cgatattgat tataaagatg acgacgacaa agttgcaacc
     1561 ctgcaggcag aaaatgttac cggtctgttt aaagattgca gcaaagttat tacaggtctg
     1621 catccgacac aggcaccgac acatctgagc gttgatacca aattcaaaac cgaaggtctg
     1681 tgtgttgata ttccgggtat tccgaaagat atgacctatc gtcgtctgat tagcatgatg
     1741 ggcttcaaaa tgaattatca ggtgaatggc tacccgaaca tgtttattac ccgtgaagaa
     1801 gcaattcgtc acgttcgtgc atggattggt tttgatgttg aaggttgtca tgcaacacgt
     1861 gaagcagttg gcaccaatct gccgctgcag ctgggtttta gcaccggtgt taatctggtt
     1921 gcagttccga ccggttatgt tgataccccg aataacaccg attttagccg tgttagcgca
     1981 aaaccgcctc cgggtgatca gtttaaacat ctgattccgc tgatgtataa aggtctgccg
     2041 tggaatgttg tgcgtatcaa aattgttcag atgctgagcg ataccctgaa aaatctgagc
     2101 gatcgtgttg tttttgttct gtgggcacat ggttttgaac tgaccagcat gaaatacttc
     2161 gtgaaaattg gtccggaacg tacctgttgt ctgtgtgatc gtcgtgcaac ctgttttagt
     2221 accgcaagcg atacctatgc atgttggcat catagcattg gcttcgatta tgtgtataac
     2281 ccgtttatga ttgatgttca gcagtggggt tttaccggca atctgcagag caatcatgat
     2341 ctgtattgtc aggttcatgg taatgcacat gttgcaagct gtgatgcaat tatgacccgt
     2401 tgtctggcag ttcatgaatg ctttgttaaa cgtgttgatt ggaccatcga atatccgatt
     2461 attggtgatg agctgaaaat taacgcagcc tgtcgtaaag ttcagcacat ggtggttaaa
     2521 gcagcactgc tggcagataa atttccggtt ctgcatgata ttggtaaccc gaaagcaatt
     2581 aaatgtgttc cgcaggcaga tgtggaatgg aaattctatg atgcacagcc gtgtagcgat
     2641 aaagcctata aaatcgaaga actgttctat agctatgcga cccatagcga caaatttacc
     2701 gatggtgttt gtctgttttg gaattgcaat gttgatcgct atccggcaaa tagcattgtt
     2761 tgccgttttg atacccgtgt tctgagcaat ctgaatctgc ctggttgtga tggtggtagc
     2821 ctgtatgtta acaaacatgc atttcatacc cctgcctttg ataaaagcgc atttgtgaat
     2881 ctgaaacagc tgccgttttt ctattatagc gatagcccgt gtgaaagcca tggtaaacag
     2941 gtggttagcg atattgatta tgttccgctg aaaagcgcaa cctgcattac ccgttgtaat
     3001 cttggtggtg cagtttgtcg tcatcatgca aatgaatatc gtctgtatct ggatgcctac
     3061 aacatgatga ttagcgcagg ttttagcctg tgggtgtata aacagtttga tacctataat
     3121 ctgtggaaca cctttacacg tctgcagtaa gctagcatga ctggtggaca gcaaatgggt
     3181 cgcggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     3241 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     3301 aggaggaact atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct
     3361 atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc
     3421 atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta
     3481 tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct
     3541 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     3601 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3661 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3721 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3781 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3841 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     3901 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     3961 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4021 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4081 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4141 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4201 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     4261 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4321 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4381 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4441 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4501 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4561 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4621 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4681 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4741 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4801 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4861 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4921 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4981 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5041 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5101 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     5161 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     5221 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     5281 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     5341 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     5401 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     5461 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     5521 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     5581 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     5641 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5701 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5761 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     5821 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     5881 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     5941 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     6001 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     6061 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     6121 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     6181 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     6241 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     6301 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     6361 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     6421 gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga
     6481 tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg
     6541 ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga
     6601 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
     6661 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac
     6721 gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg
     6781 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
     6841 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc
     6901 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc
     6961 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     7021 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     7081 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     7141 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     7201 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     7261 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     7321 ccaacgcgcg gggagaggc
//
