LOCUS       sequence-421863-        5499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NSP14 of SARS-CoV-2 with two point mutations D90A and 
            E92A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5499)
  AUTHORS   Meyer C, Garzia A, Miller MW, Huggins DJ, Myers RW, Hoffmann HH, 
            Ashbrook AW, Jannath SY, Liverton N, Kargman S, Zimmerman M, Nelson 
            AM, Sharma V, Dolgov E, Cangialosi J, Penalva-Lopez S, Alvarez N, 
            Chang CW, Oswal N, Gonzalez I, Rasheed R, Goldgirsh K, Davis JA, 
            Ramos-Espiritu L, Menezes MR, Larson C, Nitsche J, Ganichkin O, 
            Alwaseem H, Molina H, Steinbacher S, Glickman JF, Perlin DS, Rice 
            CM, Meinke PT, Tuschl T
  TITLE     Small-molecule inhibition of SARS-CoV-2 NSP14 RNA cap 
            methyltransferase.
  JOURNAL   Nature. 2024 Dec 11. doi: 10.1038/s41586-024-08320-0.
  PUBMED    39663451
REFERENCE   2  (bases 1 to 5499)
  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"; journalName: "Nature. 
            2024 Dec 11. doi: 10.1038/s41586-024-08320-0."
COMMENT     Sequence Label: pRSF_his6_sumo_SARS_CoV2_NSP14_D90A/E92A
FEATURES             Location/Qualifiers
     source          1..5499
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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..1423
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3026..3070
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3108..3126)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3122..3169
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3399..4214)
                     /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             complement(3399..4214)
                     /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     4122..4141
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4215..4306)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4322..5071)
                     /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(4332..4349)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4572..4591)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5267..5499,1..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 aaaatgtaac
     1441 aggactcttt aaagattgta gtaaggtaat cactgggtta catcctacac aggcacctac
     1501 acacctcagt gttgacacta aattcaaaac tgaaggttta tgtgttgaca tacctggcat
     1561 acctaaggac atgacctata gaagactcat ctctatgatg ggttttaaaa tgaattatca
     1621 agttaatggt taccctaaca tgtttatcac ccgcgaagaa gctataagac atgtacgtgc
     1681 atggattggc ttcgctgtcg cggggtgtca tgctactaga gaagctgttg gtaccaattt
     1741 acctttacag ctaggttttt ctacaggtgt taacctagtt gctgtaccta caggttatgt
     1801 tgatacacct aataatacag atttttccag agttagtgct aaaccaccgc ctggagatca
     1861 atttaaacac ctcataccac ttatgtacaa aggacttcct tggaatgtag tgcgtataaa
     1921 gattgtacaa atgttaagtg acacacttaa aaatctctct gacagagtcg tatttgtctt
     1981 atgggcacat ggctttgagt tgacatctat gaagtatttt gtgaaaatag gacctgagcg
     2041 cacctgttgt ctatgtgata gacgtgccac gtgcttttcc actgcttcag acacttatgc
     2101 ctgttggcat cattctattg gatttgatta cgtctataat ccgtttatga ttgatgttca
     2161 acaatggggt tttacaggta acctacaaag caaccatgat ctgtattgtc aagtccatgg
     2221 taatgcacat gtagctagtt gtgatgcaat catgactagg tgtctagctg tccacgagtg
     2281 ctttgttaag cgtgttgact ggactattga atatcctata attggtgatg aactgaagat
     2341 taatgcggct tgtagaaagg ttcaacacat ggttgttaaa gctgcattat tagcagacaa
     2401 attcccagtt cttcacgaca ttggtaaccc taaagctatt aagtgtgtac ctcaagctga
     2461 tgtagaatgg aagttctatg atgcacagcc ttgtagtgac aaagcttata aaatagaaga
     2521 attattctat tcttatgcca cacattctga caaattcaca gatggtgtat gcctattttg
     2581 gaattgcaat gtcgatagat atcctgctaa ttccattgtt tgtagatttg acactagagt
     2641 gctatctaac cttaacttgc ctggttgtga tggtggcagt ttgtatgtaa ataaacatgc
     2701 attccacaca ccagcttttg ataaaagtgc ttttgttaat ttaaaacaat taccattttt
     2761 ctattactct gacagtccat gtgagtctca tggaaaacaa gtagtgtcag atatagatta
     2821 tgtaccacta aagtctgcta cgtgtataac acgttgcaat ttaggtggtg ctgtctgtag
     2881 acatcatgct aatgagtaca gattgtatct cgatgcttat aacatgatga tctcagctgg
     2941 ctttagcttg tgggtttaca aacaatttga tacttataac ctctggaaca cttttacaag
     3001 acttcagtga ccgctcgagt ctggtaaaga aaccgctgct gcgaaatttg aacgccagca
     3061 catggactcg tctactagcg cagcttaatt aacctaggct gctgccaccg ctgagcaata
     3121 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaacctca
     3181 ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg taaaccagca
     3241 atagacataa gcggctattt aacgaccctg ccctgaaccg acgacaagct gacgaccggg
     3301 tctccgcaag tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3361 aaatacattc aaatatgtat ccgctcatga attaattctt agaaaaactc atcgagcatc
     3421 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
     3481 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
     3541 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
     3601 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
     3661 agtttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
     3721 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
     3781 cggtcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
     3841 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
     3901 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
     3961 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     4021 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     4081 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     4141 ccatataaat cagcatccat gttggaattt aatcgcggcc tagagcaaga cgtttcccgt
     4201 tgaatatggc tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4261 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg catgcagcgc
     4321 tcttccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggtg
     4381 tcagctcact caaaagcggt aatacggtta tccacagaat caggggataa agccggaaag
     4441 aacatgtgag caaaaagcaa agcaccggaa gaagccaacg ccgcaggcgt ttttccatag
     4501 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agccagaggt ggcgaaaccc
     4561 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4621 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4681 ttctcatagc tcacgctgtt ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4741 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4801 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccattg gtaactgatt
     4861 tagaggactt tgtcttgaag ttatgcacct gttaaggcta aactgaaaga acagattttg
     4921 gtgagtgcgg tcctccaacc cacttacctt ggttcaaaga gttggtagct cagcgaacct
     4981 tgagaaaacc accgttggta gcggtggttt ttctttattt atgagatgat gaatcaatcg
     5041 gtctatcaag tcaacgaaca gctattccgt tactctagat ttcagtgcaa tttatctctt
     5101 caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat
     5161 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     5221 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     5281 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     5341 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     5401 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     5461 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcg
//
