LOCUS       sequence-421866-        5882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the human RNMT-RAM complex.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5882)
  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 5882)
  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_hRNMT_RAM
FEATURES             Location/Qualifiers
     source          1..5882
                     /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
     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"
     promoter        2948..2966
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2967..2991
                     /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             3409..3453
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3491..3509)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3505..3552
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(3782..4597)
                     /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(3782..4597)
                     /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     4505..4524
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4598..4689)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4705..5454)
                     /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(4715..4732)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(4955..4974)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5650..5882,1..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 ggatccatgg caaattctgc
     1441 aaaagcagaa gaatatgaaa agatgtctct tgaacaggca aaagcgtcag tgaattctga
     1501 aacagagtct tcattcaata ttaatgaaaa cacaacagct tctgggactg ggctttctga
     1561 aaagacttct gtctgtaggc aagtagacat agcaagaaag agaaaagagt ttgaagatga
     1621 tcttgtaaag gaaagttcta gttgtgggaa agacactcca tccaagaaga gaaaacttga
     1681 tcctgaaatt gtcccagagg aaaaagattg tggtgatgct gaaggcaatt caaagaaaag
     1741 aaaaagagaa actgaggatg ttccaaaaga taaatcttct actggagatg gcactcaaaa
     1801 taagagaaaa atagcacttg aggatgttcc tgaaaagcag aaaaatctgg aagaaggaca
     1861 cagctcaaca gtggctgccc attacaatga acttcaggaa gttggtttgg agaagcgtag
     1921 tcaaagtcgt attttttacc taagaaactt taataattgg atgaaaagtg ttctcattgg
     1981 agaatttttg gaaaaggtac gacagaagaa aaaacgtgat atcactgttt tggacctggg
     2041 atgtggtaaa ggtggagatt tgctgaaatg gaaaaaagga agaattaaca agctagtttg
     2101 tactgatatt gccgatgttt ctgtcaaaca gtgtcagcag cggtatgagg acatgaaaaa
     2161 tcgtcgtgat agtgaatata ttttcagtgc agaatttata actgctgaca gctcaaagga
     2221 acttctgatt gacaaatttc gtgacccaca aatgtgtttt gacatctgca gttgtcagtt
     2281 tgtctgtcat tactcatttg agtcttatga gcaggctgac atgatgctga gaaatgcgtg
     2341 tgagagactt agccctgggg gctattttat tggtactact cccaatagct ttgaattgat
     2401 aagacgcctt gaagcttcag aaacagaatc atttggaaat gaaatatata ctgtgaaatt
     2461 tcagaagaaa ggagattatc ctttatttgg ctgcaaatat gacttcaact tggaaggtgt
     2521 tgtggatgtt cctgaattct tggtctattt tccattgcta aatgaaatgg caaagaagta
     2581 caatatgaaa ctagtctaca aaaaaacatt tctggaattc tacgaagaaa agattaagaa
     2641 caatgaaaat aaaatgctct taaaacgaat gcaggccttg gagccatatc ctgcaaatga
     2701 gagttctaaa cttgtctctg agaaggtgga tgactatgaa catgcagcaa agtacatgaa
     2761 gaacagtcaa gtaaggttac ctttgggaac cttaagtaaa tcagaatggg aagctacaag
     2821 tatttacttg gtgtttgcct ttgagaaaca gcagtgaggc gcgcctgcag gtcgacaagc
     2881 ttgcggccgc ataatgctta agtcgaacag aaagtaatcg tattgtacac ggccgcataa
     2941 tcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc cccatcttag
     3001 tatattagtt aagtataaga aggagatata catatgatga ctgacactgc cgaagctgtt
     3061 ccaaagtttg aagagatgtt tgctagtaga ttcacagaaa atgacaagga gtatcaggaa
     3121 tacctgaaac gccctcctga gtctcctcca attgttgagg aatggaatag cagagctggt
     3181 gggaaccaaa gaaacagagg caatcggttg caagacaaca gacagttcag aggcagggac
     3241 aacagatggg ggtggccaag tgacaatcga tccaatcagt ggcatggacg atcctggggt
     3301 aacaactacc cgcaacacag acaagaacct tactatcccc agcaatatgg acattatggt
     3361 tacaaccagc ggcctcctta cggttactac tgaccgctcg agtctggtaa agaaaccgct
     3421 gctgcgaaat ttgaacgcca gcacatggac tcgtctacta gcgcagctta attaacctag
     3481 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     3541 aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact gcttccggta
     3601 gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc ctgccctgaa
     3661 ccgacgacaa gctgacgacc gggtctccgc aagtggcact tttcggggaa atgtgcgcgg
     3721 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     3781 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     3841 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     3901 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     3961 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     4021 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     4081 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     4141 gcgcctgagc gagacgaaat acgcggtcgc tgttaaaagg acaattacaa acaggaatcg
     4201 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     4261 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     4321 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     4381 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     4441 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     4501 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     4561 gcctagagca agacgtttcc cgttgaatat ggctcatact cttccttttt caatattatt
     4621 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4681 ataaacaaat aggcatgcag cgctcttccg cttcctcgct cactgactcg ctacgctcgg
     4741 tcgttcgact gcggcgagcg gtgtcagctc actcaaaagc ggtaatacgg ttatccacag
     4801 aatcagggga taaagccgga aagaacatgt gagcaaaaag caaagcaccg gaagaagcca
     4861 acgccgcagg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4921 tcaagccaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4981 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     5041 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gttggtatct cagttcggtg
     5101 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     5161 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     5221 gcagcagcca ttggtaactg atttagagga ctttgtcttg aagttatgca cctgttaagg
     5281 ctaaactgaa agaacagatt ttggtgagtg cggtcctcca acccacttac cttggttcaa
     5341 agagttggta gctcagcgaa ccttgagaaa accaccgttg gtagcggtgg tttttcttta
     5401 tttatgagat gatgaatcaa tcggtctatc aagtcaacga acagctattc cgttactcta
     5461 gatttcagtg caatttatct cttcaaatgt agcacctgaa gtcagcccca tacgatataa
     5521 gttgtaattc tcatgttagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     5581 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     5641 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5701 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     5761 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5821 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     5881 cg
//
