LOCUS       sequence-333997-        4882 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express His-SUMO-nsp8 (SARS-CoV-2) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4882)
  AUTHORS   Bertolin AP, Weissmann F, Zeng J, Posse V, Milligan JC, Canal B, 
            Ulferts R, Wu M, Drury LS, Howell M, Beale R, Diffley JFX
  TITLE     Identifying SARS-CoV-2 antiviral compounds by screening for small 
            molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase.
  JOURNAL   Biochem J. 2021 Jul 16;478(13):2425-2443. doi: 10.1042/BCJ20210200.
  PUBMED    34198323
REFERENCE   2  (bases 1 to 4882)
  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"; doi: "10"; journalName: 
            "Biochem J"; date: "2021-07-16- 16"; volume: "478"; issue: "13"; 
            pages: "2425-2443"
COMMENT     Sequence Label: pK27Sumo_His-SUMO-nsp8 (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..4882
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(589..675)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(894..1709)
                     /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(894..1709)
                     /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     1617..1636
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1701..1720
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     terminator      complement(1768..1862)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(2506..2796)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     RBS             2881..2892
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    2911..2927
                     /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)."
     promoter        complement(join(2935..2941,2942..2958,2959..2964,
                     2965..2979))
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    2943..2959
                     /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)."
     primer_bind     complement(3003..3021)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      3112..3700
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3601..3620
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3854..3871
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3886..4026
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3952..3971)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4112..4134
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        4218..4295
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             join(4296..4882,1..496)
                     /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"
     primer_bind     complement(4315..4334)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg
       61 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg
      121 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc
      181 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac
      241 cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct
      301 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa
      361 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggccccca atacgcaaac
      421 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
      481 ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcacaat
      541 tctcatgttt gacagcttat catcgactgc acgtaatcta gagggcggat ttgtcctact
      601 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc
      661 ctttcgtttt atttgatgcc tcaagctaga gagtcattac cccaggcgtt taagggcacc
      721 aataactgcc ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt
      781 cattaagcat tctgccgaca tggaagccat cacaaacggc atgatgaacc tgaatcgcca
      841 gcggcatcag caccttgtcg ccttgcgtat aatatttgcc catgaattaa ttcttagaaa
      901 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
      961 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     1021 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     1081 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     1141 ggtgagaatg gcaaaagttt gtgcatttct ttccagactt gttcaacagg ccagccatta
     1201 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     1261 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     1321 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     1381 aatacctgga atgctgtttt gccggggatc gcagtcgtga gtaaccaagc atcatcagga
     1441 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     1501 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     1561 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     1621 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     1681 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     1741 gacagtttat tttcatggct agcttggatt ctcaccaata aaaaacgccc ggcggcaacc
     1801 gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag
     1861 tccaagctca gctaattaag cttagtgatg gtgatgtgcg gccgcttatt actgcagttt
     1921 aactgcgcta tttgcacgca gtgcggtaac aatcagaggc catgccagat tcgggctatt
     1981 atccatgcta atttcgctca gctgcacaat tttgctatct gcatcaacaa cctgctgaat
     2041 ttcccacagt gcgcttgcat aggtaaaggt ggtgccatca caggtgtttt tataggtgtt
     2101 gtaatccgga ataaccacca tcagttttgc tgcggtggtc agcggaataa tgttcagcgg
     2161 aacacaacca tcacgggcat tattgataat gttgttcagg gcatcattat ccagtttgcg
     2221 cagcatggta aacagcatgg tctgcattgc gctggtaact tttgcacgtt tatcttcgct
     2281 acgtgcctgt ttatacatct gggtcattgc ctgatctgcc atcttttcca gtttacgctg
     2341 cattgctgca tcacgatcaa attcgctttt ggccacgttc agtgactttt tcagtttttt
     2401 cagaacaact tcgctatcac cattggcaac tgcctgttcg tatgcttctt gtgcggttgc
     2461 aaatgctgca tagctcggca ggctgctaaa ttcgctggca attgcgccac caatctgttc
     2521 tctgtgagcc tcaataatat cgttatcctc catgtccaaa tcttcagggg tctgatcagc
     2581 ttgaattcta ataccgtcgt acaagaatct taaggagtcc atttccttac cctgtctttt
     2641 agcgaacgct tccatcagcc ttcttaaagg agtggtcttt ttgatcttga agaagatctc
     2701 tgaagatcca tcggacacct ttaaattgat gtgagtctca ggcttgactt ctggcttgac
     2761 ctctggctta gcttcttgat tgacttctga gtccgatccg gtatggtgat gactgcctga
     2821 atgatggtgg tgtccggtat ggtgatggcc tgaatgatgg tgatgcttgc tcatggttaa
     2881 tttctcctct ttaatgaatt ctgtgtgaaa ttgttatccg ctcacaattg aatctattat
     2941 aattgttatc cgctcacaaa gcaaataaat tttttatgat ttctcgaggt gaagacgaaa
     3001 gggcctcgtg atacgcctat ttttataggt taatgtcatg accaaaatcc cttaacgtga
     3061 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3121 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3181 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3241 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3301 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     3361 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     3421 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     3481 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     3541 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     3601 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3661 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3721 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3781 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3841 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     3901 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     3961 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4021 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4081 gagacggtca cagcttgtct gtaagcggat gccgggagct gcatgtgtca gaggttttca
     4141 ccgtcatcac cgaaacgcgc gaggcagctc tagatcatgc gcacccgtgg ccaggaccca
     4201 acgctgcccg aaattccgac accatcgaat ggtgcaaaac ctttcgcggt atggcatgat
     4261 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     4321 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     4381 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     4441 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     4501 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     4561 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     4621 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     4681 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     4741 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     4801 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     4861 taagttctgt ctcggcgcgt ct
//
