LOCUS       sequence-421665-        5469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NSP14 of hCoV 229E.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5469)
  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 5469)
  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_229E_NSP14
FEATURES             Location/Qualifiers
     source          1..5469
                     /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             2996..3040
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3078..3096)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3092..3139
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3369..4184)
                     /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(3369..4184)
                     /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     4092..4111
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4185..4276)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4292..5041)
                     /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(4302..4319)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4542..4561)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5237..5469,1..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 ggatccgaaa gcagttgtgg
     1441 tttgtttaag gattgtgcac gtaaccctat tgatttacca ccaagtcatg ccactactta
     1501 tttgtcattg tctgatagat ttaagactag tggtgacttg gctgttcaaa taggtaacaa
     1561 caatgtttgt acctatgaac atgtgatttc atatatgggt ttcaggtttg atgttagcat
     1621 gcctggtagt catagtttgt tctgtactag agactttgcc atgcgtcatg tcagaggttg
     1681 gttaggaatg gatgtggaag gtgcacatgt cacaggtgac aatgttggca ctaatgtacc
     1741 tctacaagtt ggtttttcca atggtgttga ttttgtagct caacctgaag gttgtgttct
     1801 aacaaacact ggcagtgttg taaaacctgt tcgtgctcgt gcaccacctg gagaacaatt
     1861 cactcacatt gtacctctgt tacgcaaggg acaaccttgg agtgtgttga gaaaacgtat
     1921 tgttcaaatg atagcagatt ttcttgctgg ctcatctgat gtactggtgt ttgtactttg
     1981 ggctggcggt ttagagttga ccactatgcg ttattttgtt aagattggag ctgttaaaca
     2041 ttgccaatgt ggtactgttg caacatgcta caattctgtt agtaatgact attgttgctt
     2101 taaacatgca ttgggctgtg actatgttta taatccatat gtcatagata ttcaacaatg
     2161 gggttatgtt ggttcactct ccactaatca ccatgcaatt tgtaatgttc atagaaatga
     2221 gcatgttgct tctggtgatg ctattatgac tagatgtttg gctgtgtatg actgctttgt
     2281 taagaatgtg gattggtcaa ttacctaccc tatgatagct aatgaaaatg ccataaacaa
     2341 gggcggtcgc actgtgcaga gtcatattat gcgtgctgct attaaattgt acaaccctaa
     2401 agcaatccat gacattggta atcctaaggg tattcgttgt gctgtaactg atgccaagtg
     2461 gtattgttat gacaagaacc ctattaattc taatgtgaaa acattggagt atgattacat
     2521 gacacatggc caaatggatg gcttgtgttt gttttggaat tgtaatgtgg atatgtaccc
     2581 tgaattctca attgtttgca ggtttgacac acgtacacga tctacattga accttgaagg
     2641 tgtaaatggt gggtcattgt atgtcaataa tcatgcattt cacactcctg cttatgataa
     2701 acgtgctatg gctaaattga aaccagcacc gtttttctac tatgacgacg gttcatgtga
     2761 ggttgttcac gatcaagtta actatgttcc tttgagagcc actaattgca ttaccaagtg
     2821 taatattggt ggtgctgtat gttctaagca cgctaatctc tatagagcat atgttgagtc
     2881 atataacatt tttactcaag ctggttttaa tatttgggtt cctaccacgt ttgattgtta
     2941 taatttgtgg cagacattca cagatgtcaa tttacaatga ccgctcgagt ctggtaaaga
     3001 aaccgctgct gcgaaatttg aacgccagca catggactcg tctactagcg cagcttaatt
     3061 aacctaggct gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg
     3121 ggtcttgagg ggttttttgc tgaaacctca ggcatttgag aagcacacgg tcacactgct
     3181 tccggtagtc aataaaccgg taaaccagca atagacataa gcggctattt aacgaccctg
     3241 ccctgaaccg acgacaagct gacgaccggg tctccgcaag tggcactttt cggggaaatg
     3301 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3361 attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga
     3421 ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg
     3481 cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca
     3541 atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga
     3601 gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca
     3661 acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt
     3721 cgtgattgcg cctgagcgag acgaaatacg cggtcgctgt taaaaggaca attacaaaca
     3781 ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa
     3841 tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac
     3901 catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc
     3961 agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt
     4021 ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat
     4081 tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt
     4141 aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcatactctt cctttttcaa
     4201 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4261 tagaaaaata aacaaatagg catgcagcgc tcttccgctt cctcgctcac tgactcgcta
     4321 cgctcggtcg ttcgactgcg gcgagcggtg tcagctcact caaaagcggt aatacggtta
     4381 tccacagaat caggggataa agccggaaag aacatgtgag caaaaagcaa agcaccggaa
     4441 gaagccaacg ccgcaggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4501 tcgacgctca agccagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4561 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4621 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgtt ggtatctcag
     4681 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4741 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4801 gccactggca gcagccattg gtaactgatt tagaggactt tgtcttgaag ttatgcacct
     4861 gttaaggcta aactgaaaga acagattttg gtgagtgcgg tcctccaacc cacttacctt
     4921 ggttcaaaga gttggtagct cagcgaacct tgagaaaacc accgttggta gcggtggttt
     4981 ttctttattt atgagatgat gaatcaatcg gtctatcaag tcaacgaaca gctattccgt
     5041 tactctagat ttcagtgcaa tttatctctt caaatgtagc acctgaagtc agccccatac
     5101 gatataagtt gtaattctca tgttagtcat gccccgcgcc caccggaagg agctgactgg
     5161 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     5221 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     5281 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     5341 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     5401 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     5461 ttaacggcg
//
