LOCUS Exported 6302 bp ds-DNA circular SYN 03-JUN-2021 DEFINITION To express nsp14-nsp10 fusion protein in E. coli. ACCESSION . VERSION . KEYWORDS pK27Sumo_His-SUMO-nsp14-nsp10 fusion (SARS-CoV-2) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6302) AUTHORS Canal B, McClure AW, Curran JF, Wu M, Ulferts R, Weissmann F, Zeng J, Bertolin AP, Milligan JC, Basu S, Drury LS, Deegan T, 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 bioRxiv https://www.biorxiv.org/content/10.1101/2021.04.07.438812v1 REFERENCE 2 (bases 1 to 6302) AUTHORS . TITLE Direct Submission JOURNAL Exported Jun 3, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6302 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 864..883 /label=LacI-R /note="LacI, reverse primer" promoter complement(903..980) /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(1064..1086) /label=pGEX 3' /note="pGEX vectors, reverse primer" misc_feature 1172..1312 /label=bom /note="basis of mobility region from pBR322" primer_bind 1227..1246 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind complement(1327..1344) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1498..2086) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1578..1597) /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2177..2195 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 2219..2263 /label=T5 promoter /note="bacteriophage T5 promoter for E. coli RNA polymerase, with embedded lac operator" protein_bind 2239..2255 /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 2271..2287 /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 2306..2317 /note="strong bacterial ribosome binding site (Elowitz and Leibler, 2000)" CDS 2402..2689 /codon_start=1 /gene="S. cerevisiae SMT3 (truncated)" /product="cleavable ubiquitin-like protein tag" /label=SUMO /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG" terminator 4756..4850 /label=lambda t0 terminator /note="transcription terminator from phage lambda" primer_bind complement(4898..4917) /label=pENTR-R /note="pENTR vectors, reverse primer" CDS 4909..5724 /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(4982..5001) /label=Kan-R /note="Kanamycin resistance gene, reverse primer" terminator 5943..6029 /gene="Escherichia coli rrnB" /label=rrnB T1 terminator /note="transcription terminator T1 from the E. coli rrnB gene" CDS complement(6122..902) /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 acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa 61 catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc 121 ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc 181 gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca 241 ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc 301 cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt 361 tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag 421 aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta 481 gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc 541 ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt 601 cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc 661 gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac 721 gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc 781 gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg 841 gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc 901 acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt 961 ttgcaccatt cgatggtgtc ggaatttcgg gcagcgttgg gtcctggcca cgggtgcgca 1021 tgatctagag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc 1081 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagcagacaa gcccgtcagg 1141 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata 1201 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca 1261 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc 1321 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc 1381 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat 1441 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 1501 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 1561 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 1621 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 1681 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 1741 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 1801 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 1861 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 1921 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt 1981 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 2041 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 2101 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 2161 gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtcttcacc tcgagaaatc 2221 ataaaaaatt tatttgcttt gtgagcggat aacaattata atagattcaa ttgtgagcgg 2281 ataacaattt cacacagaat tcattaaaga ggagaaatta accatgagca agcatcacca 2341 tcattcaggc catcaccata ccggacacca ccatcattca ggcagtcatc accataccgg 2401 atcggactca gaagtcaatc aagaagctaa gccagaggtc aagccagaag tcaagcctga 2461 gactcacatc aatttaaagg tgtccgatgg atcttcagag atcttcttca agatcaaaaa 2521 gaccactcct ttaagaaggc tgatggaagc gttcgctaaa agacagggta aggaaatgga 2581 ctccttaaga ttcttgtacg acggtattag aattcaagct gatcagaccc ctgaagattt 2641 ggacatggag gataacgata ttattgaggc tcacagagaa cagattggtg gcgcagaaaa 2701 tgttaccggt ctgtttaaag attgcagcaa agttattaca ggtctgcatc cgacacaggc 2761 accgacacat ctgagcgttg ataccaaatt caaaaccgaa ggtctgtgtg ttgatattcc 2821 gggtattccg aaagatatga cctatcgtcg tctgattagc atgatgggct tcaaaatgaa 2881 ttatcaggtg aatggctacc cgaacatgtt tattacccgt gaagaagcaa ttcgtcacgt 2941 tcgtgcatgg attggttttg atgttgaagg ttgtcatgca acacgtgaag cagttggcac 3001 caatctgccg ctgcagctgg gttttagcac cggtgttaat ctggttgcag ttccgaccgg 3061 ttatgttgat accccgaata acaccgattt tagccgtgtt agcgcaaaac cgcctccggg 3121 tgatcagttt aaacatctga ttccgctgat gtataaaggt ctgccgtgga atgttgtgcg 3181 tatcaaaatt gttcagatgc tgagcgatac cctgaaaaat ctgagcgatc gtgttgtttt 3241 tgttctgtgg gcacatggtt ttgaactgac cagcatgaaa tacttcgtga aaattggtcc 3301 ggaacgtacc tgttgtctgt gtgatcgtcg tgcaacctgt tttagtaccg caagcgatac 3361 ctatgcatgt tggcatcata gcattggctt cgattatgtg tataacccgt ttatgattga 3421 tgttcagcag tggggtttta ccggcaatct gcagagcaat catgatctgt attgtcaggt 3481 tcatggtaat gcacatgttg caagctgtga tgcaattatg acccgttgtc tggcagttca 3541 tgaatgcttt gttaaacgtg ttgattggac catcgaatat ccgattattg gtgatgagct 3601 gaaaattaac gcagcctgtc gtaaagttca gcacatggtg gttaaagcag cactgctggc 3661 agataaattt ccggttctgc atgatattgg taacccgaaa gcaattaaat gtgttccgca 3721 ggcagatgtg gaatggaaat tctatgatgc acagccgtgt agcgataaag cctataaaat 3781 cgaagaactg ttctatagct atgcgaccca tagcgacaaa tttaccgatg gtgtttgtct 3841 gttttggaat tgcaatgttg atcgctatcc ggcaaatagc attgtttgcc gttttgatac 3901 ccgtgttctg agcaatctga atctgcctgg ttgtgatggt ggtagcctgt atgttaacaa 3961 acatgcattt catacccctg cctttgataa aagcgcattt gtgaatctga aacagctgcc 4021 gtttttctat tatagcgata gcccgtgtga aagccatggt aaacaggtgg ttagcgatat 4081 tgattatgtt ccgctgaaaa gcgcaacctg cattacccgt tgtaatcttg gtggtgcagt 4141 ttgtcgtcat catgcaaatg aatatcgtct gtatctggat gcctacaaca tgatgattag 4201 cgcaggtttt agcctgtggg tgtataaaca gtttgatacc tataatctgt ggaacacctt 4261 tacacgtctg caggggtagc ggtggtagtg caggtaatgc aaccgaagtt ccggcaaata 4321 gcaccgttct gagcttttgt gcatttgcag ttgatgcagc aaaagcctat aaagactatc 4381 tggcaagcgg tggtcagccg attaccaatt gtgttaaaat gctgtgtacc cataccggca 4441 ccggtcaggc aattaccgtt acaccggaag caaatatgga tcaagaaagc tttggtggtg 4501 caagctgttg tctgtattgt cgttgtcata ttgatcaccc gaatccgaaa ggtttttgtg 4561 atctgaaagg taagtatgtg cagattccga ccacctgtgc aaatgatccg gttggtttta 4621 ccctgaaaaa taccgtttgc accgtttgtg gtatgtggaa aggttatggt tgtagctgtg 4681 atcagctgcg tgaaccgatg ctgcagtaat aagcggccgc acatcaccat cactaagctt 4741 aattagctga gcttggactc ctgttgatag atccagtaat gacctcagaa ctccatctgg 4801 atttgttcag aacgctcggt tgccgccggg cgttttttat tggtgagaat ccaagctagc 4861 catgaaaata aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt 4921 caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat 4981 gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat 5041 gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat 5101 gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc 5161 aagcatttta tccgtactcc tgatgatgct tggttactca cgactgcgat ccccggcaaa 5221 acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg 5281 gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat 5341 cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt 5401 gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcacaaa 5461 cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt 5521 atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac 5581 cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag 5641 aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat 5701 ttgatgctcg atgagttttt ctaagaatta attcatgggc aaatattata cgcaaggcga 5761 caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt 5821 cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg gggcgtaatt 5881 tttttaaggc agttattggt gcccttaaac gcctggggta atgactctct agcttgaggc 5941 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt 6001 cggtgaacgc tctcctgagt aggacaaatc cgccctctag attacgtgca gtcgatgata 6061 agctgtcaaa catgagaatt gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg 6121 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 6181 acgcgcgggg agaggcggtt tgcgtattgg gggccagggt ggtttttctt ttcaccagtg 6241 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt 6301 cc //