LOCUS Exported 6711 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION The SARS-CoV-2 NSP12 coding sequence was cloned into a modified pRSFDuet-1 vector (Novagen) bearing an N-terminal His6-SUMO-tag which is cleavable by the ubiquitin-like protease (ULP1).. ACCESSION . VERSION . KEYWORDS pRSFDuet-sumo-NSP12 (SARS-CoV-2) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6711) AUTHORS Chen J, Malone B, Llewellyn E, Grasso M, Shelton PMM, Olinares PDB, Maruthi K, Eng ET, Vatandaslar H, Chait B, Kapoor T, Darst SA, Campbell EA TITLE Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex. JOURNAL bioRxiv. 2020 Jul 13. doi: 10.1101/2020.07.08.194084. PUBMED 32676607 REFERENCE 2 (bases 1 to 6711) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6711 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 812..831 /label=LacI-R /note="LacI, reverse primer" promoter complement(851..928) /gene="lacI" /label=lacI promoter 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)." CDS 1073..1090 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1100..1117 /codon_start=1 /product="thrombin recognition and cleavage site" /label=thrombin site /translation="LVPRGS" CDS 1130..1420 /codon_start=1 /gene="S. cerevisiae SMT3 (truncated)" /product="cleavable ubiquitin-like protein tag" /label=SUMO /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG" CDS 4238..4282 /codon_start=1 /product="affinity and epitope tag derived from pancreatic ribonuclease A" /label=S-Tag /translation="KETAAAKFERQHMDS" primer_bind complement(4320..4338) /label=T7 Term /note="T7 terminator, reverse primer" terminator 4334..4381 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" CDS complement(4611..5426) /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 5334..5353 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" promoter complement(5427..5518) /gene="bla" /label=AmpR promoter rep_origin complement(5534..6283) /direction=LEFT /label=RSF ori /note="Plasmids containing the RSF 1030 origin of replication can be propagated in E. coli cells that contain additional plasmids with compatible origins." primer_bind complement(5544..5561) /label=L4440 /note="L4440 vector, forward primer" primer_bind complement(5784..5803) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(6479..850) /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 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa 61 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa 121 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg 181 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat 241 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca 301 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt 361 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg 421 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa 481 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga 541 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt 601 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa 661 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct 721 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca 781 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta 841 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc 901 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc 961 tgcattagga aattaatacg actcactata ggggaattgt gagcggataa caattcccct 1021 gtagaaataa ttttgtttaa ctttaataag gagatatacc atgggcagca gccatcatca 1081 tcatcatcac agcagcggcc tggtgccgcg cggcagccac atggcaagca tgtcggactc 1141 agaagtcaat caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat 1201 caatttaaag gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc 1261 tttaagaagg ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag 1321 attcttgtac gacggtatta gaatccaagc tgatcagacc cctgaagatt tggacatgga 1381 ggataacgat attattgagg ctcacagaga acagattggt ggatccgctg atgcacaatc 1441 gtttttaaac cgggtttgcg gtgtaagtgc agcccgtctt acaccgtgcg gcacaggcac 1501 tagtactgat gtcgtataca gggcttttga catctacaat gataaagtag ctggttttgc 1561 taaattccta aaaactaatt gttgtcgctt ccaagaaaag gacgaagatg acaatttaat 1621 tgattcttac tttgtagtta agagacacac tttctctaac taccaacatg aagaaacaat 1681 ttataattta cttaaggatt gtccagctgt tgctaaacat gacttcttta agtttagaat 1741 agacggtgac atggtaccac atatatcacg tcaacgtctt actaaataca caatggcaga 1801 cctcgtctat gctttaaggc attttgatga aggtaattgt gacacattaa aagaaatact 1861 tgtcacatac aattgttgtg atgatgatta tttcaataaa aaggactggt atgattttgt 1921 agaaaaccca gatatattac gcgtatacgc caacttaggt gaacgtgtac gccaagcttt 1981 gttaaaaaca gtacaattct gtgatgccat gcgaaatgct ggtattgttg gtgtactgac 2041 attagataat caagatctca atggtaactg gtatgatttc ggtgatttca tacaaaccac 2101 gccaggtagt ggagttcctg ttgtagattc ttattattca ttgttaatgc ctatattaac 2161 cttgaccagg gctttaactg cagagtcaca tgttgacact gacttaacaa agccttacat 2221 taagtgggat ttgttaaaat atgacttcac ggaagagagg ttaaaactct ttgaccgtta 2281 ttttaaatat tgggatcaga cataccaccc aaattgtgtt aactgtttgg atgacagatg 2341 cattctgcat tgtgcaaact ttaatgtttt attctctaca gtgttcccac ctacaagttt 2401 tggaccacta gtgagaaaaa tatttgttga tggtgttcca tttgtagttt caactggata 2461 ccacttcaga gagctaggtg ttgtacataa tcaggatgta aacttacata gctctagact 2521 tagttttaag gaattacttg tgtatgctgc tgaccctgct atgcacgctg cttctggtaa 2581 tctattacta gataaacgca ctacgtgctt ttcagtagct gcacttacta acaatgttgc 2641 ttttcaaact gtcaaacccg gtaattttaa caaagacttc tatgactttg ctgtgtctaa 2701 gggtttcttt aaggaaggaa gttctgttga attaaaacac ttcttctttg ctcaggatgg 2761 taatgctgct atcagcgatt atgactacta tcgttataat ctaccaacaa tgtgtgatat 2821 cagacaacta ctatttgtag ttgaagttgt tgataagtac tttgattgtt acgatggtgg 2881 ctgtattaat gctaaccaag tcatcgtcaa caacctagac aaatcagctg gttttccatt 2941 taataaatgg ggtaaggcta gactttatta tgattcaatg agttatgagg atcaagatgc 3001 acttttcgca tatacaaaac gtaatgtcat ccctactata actcaaatga atcttaagta 3061 tgccattagt gcaaagaata gagctcgcac cgtagctggt gtctctatct gtagtactat 3121 gaccaataga cagtttcatc aaaaattatt gaaatcaata gccgccacta gaggagctac 3181 tgtagtaatt ggaacaagca aattctatgg tggttggcac aacatgttaa aaactgttta 3241 tagtgatgta gaaaaccctc accttatggg ttgggattat cctaaatgtg atagagccat 3301 gcctaacatg cttagaatta tggcctcact tgttcttgct cgcaaacata caacgtgttg 3361 tagcttgtca caccgtttct atagattagc taatgagtgt gctcaagtat tgagtgaaat 3421 ggtcatgtgt ggcggttcac tatatgttaa accaggtgga acctcatcag gagatgccac 3481 aactgcttat gctaatagtg tttttaacat ttgtcaagct gtcacggcca atgttaatgc 3541 acttttatct actgatggta acaaaattgc cgataagtat gtccgcaatt tacaacacag 3601 actttatgag tgtctctata gaaatagaga tgttgacaca gactttgtga atgagtttta 3661 cgcatatttg cgtaaacatt tctcaatgat gatactctct gacgatgctg ttgtgtgttt 3721 caatagcact tatgcatctc aaggtctagt ggctagcata aagaacttta agtcagttct 3781 ttattatcaa aacaatgttt ttatgtctga agcaaaatgt tggactgaga ctgaccttac 3841 taaaggacct catgaatttt gctctcaaca tacaatgcta gttaaacagg gtgatgatta 3901 tgtgtacctt ccttacccag atccatcaag aatcctaggg gccggctgtt ttgtagatga 3961 tatcgtaaaa acagatggta cacttatgat tgaacggttc gtgtctttag ctatagatgc 4021 ttacccactt actaaacatc ctaatcagga gtatgctgat gtctttcatt tgtacttaca 4081 atacataaga aagctacatg atgagttaac aggacacatg ttagacatgt attctgttat 4141 gcttactaat gataacactt caaggtattg ggaacctgag ttttatgagg ctatgtacac 4201 accgcataca gtcttacagt gaccgctcga gtctggtaaa gaaaccgctg ctgcgaaatt 4261 tgaacgccag cacatggact cgtctactag cgcagcttaa ttaacctagg ctgctgccac 4321 cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt 4381 gctgaaacct caggcatttg agaagcacac ggtcacactg cttccggtag tcaataaacc 4441 ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgacaag 4501 ctgacgaccg ggtctccgca agtggcactt ttcggggaaa tgtgcgcgga acccctattt 4561 gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac 4621 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt 4681 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca 4741 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc 4801 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt 4861 gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc 4921 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg 4981 agacgaaata cgcggtcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg 5041 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat 5101 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta 5161 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc 5221 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc 5281 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga 5341 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa 5401 gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg aagcatttat 5461 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 5521 ggcatgcagc gctcttccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg 5581 cggcgagcgg tgtcagctca ctcaaaagcg gtaatacggt tatccacaga atcaggggat 5641 aaagccggaa agaacatgtg agcaaaaagc aaagcaccgg aagaagccaa cgccgcaggc 5701 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagccagag 5761 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 5821 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 5881 aagcgtggcg ctttctcata gctcacgctg ttggtatctc agttcggtgt aggtcgttcg 5941 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 6001 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccat 6061 tggtaactga tttagaggac tttgtcttga agttatgcac ctgttaaggc taaactgaaa 6121 gaacagattt tggtgagtgc ggtcctccaa cccacttacc ttggttcaaa gagttggtag 6181 ctcagcgaac cttgagaaaa ccaccgttgg tagcggtggt ttttctttat ttatgagatg 6241 atgaatcaat cggtctatca agtcaacgaa cagctattcc gttactctag atttcagtgc 6301 aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag ttgtaattct 6361 catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg 6421 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc 6481 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg 6541 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga 6601 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca 6661 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc g //