LOCUS       sequence-421663-        5481 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NSP14 of hCoV OC43.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5481)
  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 5481)
  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_OC43_NSP14
FEATURES             Location/Qualifiers
     source          1..5481
                     /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             3008..3052
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3090..3108)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3104..3151
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3381..4196)
                     /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(3381..4196)
                     /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     4104..4123
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4197..4288)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4304..5053)
                     /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(4314..4331)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4554..4573)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5249..5481,1..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 ggatcctgta gtactaattt
     1441 atttaaagat tgtagcaaga gttatagcgg ttatcaccca gctcatgctc cttcattttt
     1501 ggcagtagat gacaaatata aggcaactgg cgatttagcc gtgtgtcttg gtattggtga
     1561 ttctgctgtt acatattcaa gattaatatc actcatgggt tttaaattgg atgttaccct
     1621 tgatgggtat tgtaagcttt ttataactaa agaagaagct gttaaacgcg tgcgtgcctg
     1681 ggttggcttt gatgctgaag gtgctcatgc cacgcgtgat agcattggga caaatttccc
     1741 acttcaatta ggattttcca caggaattga ttttgttgtg gaagccactg gtttgtttgc
     1801 tgatagagat ggttacagct ttaaaaaggc tgtggcgaaa gctcctcctg gtgaacaatt
     1861 taagcacctc atccctttga tgacgagagg tcatcgctgg gctgttgtta gacctagaat
     1921 agtacaaatg tttgcagatc atttaattga tctgtctgat tgtgttgtgc tagttacatg
     1981 ggcagccaac tttgagctca cttgtctccg ctactttgca aaagtagggc gtgagatttc
     2041 ttgtaatgta tgcactaaac gtgccacagt ttacaattct agaactagtt actatggttg
     2101 ttggcgccat agtgttacat gtgattactt gtataatcca cttattgttg atattcaaca
     2161 gtggggatat attggttctt tatcaagtaa tcatgattta tattgtagtg tccataaagg
     2221 agcacatgtt gcttcctctg atgctataat gacacggtgt ttggccgttt atgattgctt
     2281 ttgcaataat attaattgga atgtggagta tcccatcatt tcaaatgagt taagtattaa
     2341 tacctcttgt agggtcttgc agcgtgtgat tcttaaagct gccatgctct gcaacagata
     2401 tactttgtgt tatgatattg gcaacccaaa agcgattgcc tgtgtcaaag attttgattt
     2461 taagttctat gatgcccaac caattgttaa gtctgttaag actcttttgt attcttttga
     2521 ggcacataag gactctttta aagacggttt gtgtatgttt tggaactgta atgtggataa
     2581 gtatccaccg aatgcagttg tatgtagatt tgacactaga gtgttgaata atttaaatct
     2641 tcctggctgt aatggaggta gtttgtatgt taataaacat gcattccaca ctaaaccctt
     2701 tgctagggca gcctttgagc atttgaagcc tatgccattc ttctattatt cagatacgcc
     2761 ttgtgtgtat atggatggca tggatgctaa gcaggttgat tatgtacctt tgaaatctgc
     2821 cacgtgcatc acaagatgca atttaggtgg tgcagtttgt ttaaaacatg ctgaagagta
     2881 tcgtgagtac ttagagtctt acaatacagc tactacagca ggttttactt tttgggtcta
     2941 taagacattt gatttttata atttgtggaa tacgttcacc aagctacaat gaccgctcga
     3001 gtctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag cacatggact cgtctactag
     3061 cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     3121 ggcctctaaa cgggtcttga ggggtttttt gctgaaacct caggcatttg agaagcacac
     3181 ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat
     3241 ttaacgaccc tgccctgaac cgacgacaag ctgacgaccg ggtctccgca agtggcactt
     3301 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3361 atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     3421 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     3481 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     3541 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     3601 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg catttctttc
     3661 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     3721 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcggtcgct gttaaaagga
     3781 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     3841 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     3901 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     3961 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     4021 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     4081 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     4141 atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg gctcatactc
     4201 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4261 tttgaatgta tttagaaaaa taaacaaata ggcatgcagc gctcttccgc ttcctcgctc
     4321 actgactcgc tacgctcggt cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg
     4381 gtaatacggt tatccacaga atcaggggat aaagccggaa agaacatgtg agcaaaaagc
     4441 aaagcaccgg aagaagccaa cgccgcaggc gtttttccat aggctccgcc cccctgacga
     4501 gcatcacaaa aatcgacgct caagccagag gtggcgaaac ccgacaggac tataaagata
     4561 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4621 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4681 ttggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4741 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4801 acacgactta tcgccactgg cagcagccat tggtaactga tttagaggac tttgtcttga
     4861 agttatgcac ctgttaaggc taaactgaaa gaacagattt tggtgagtgc ggtcctccaa
     4921 cccacttacc ttggttcaaa gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg
     4981 tagcggtggt ttttctttat ttatgagatg atgaatcaat cggtctatca agtcaacgaa
     5041 cagctattcc gttactctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag
     5101 tcagccccat acgatataag ttgtaattct catgttagtc atgccccgcg cccaccggaa
     5161 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5221 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5281 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5341 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5401 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     5461 gtttgatggt ggttaacggc g
//
