LOCUS       sequence-333987-        4537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express His-SUMO-nsp7 (SARS-CoV-2) in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4537)
  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 4537)
  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-nsp7 (SARS-CoV-2)
FEATURES             Location/Qualifiers
     source          1..4537
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(45..1127)
                     /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     1089..1108
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1128..1205)
                     /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."
     primer_bind     complement(1289..1311)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1397..1537
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1452..1471
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1552..1569)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1723..2311)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1803..1822)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2402..2420
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2444..2458,2459..2464,2465..2481,2482..2488)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    2464..2480
                     /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)."
     protein_bind    2496..2512
                     /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)."
     RBS             2531..2542
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             2627..2917
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR
                     RLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     terminator      3216..3310
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(3358..3377)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             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             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     complement(3442..3461)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      4403..4489
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 attgtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
       61 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
      121 gtttgcgtat tgggggccag ggtggttttt cttttcacca gtgagacggg caacagctga
      181 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
      241 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
      301 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
      361 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
      421 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
      481 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
      541 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
      601 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      661 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      721 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      781 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      841 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      901 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      961 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     1021 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     1081 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     1141 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg
     1201 gtgtcggaat ttcgggcagc gttgggtcct ggccacgggt gcgcatgatc tagagctgcc
     1261 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gcatccgctt
     1321 acagacaagc tgtgaccgtc tccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     1381 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     1441 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     1501 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     1561 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     1621 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1681 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1741 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1801 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1861 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1921 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1981 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     2041 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     2101 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     2161 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     2221 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2281 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2341 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgacat taacctataa
     2401 aaataggcgt atcacgaggc cctttcgtct tcacctcgag aaatcataaa aaatttattt
     2461 gctttgtgag cggataacaa ttataataga ttcaattgtg agcggataac aatttcacac
     2521 agaattcatt aaagaggaga aattaaccat gagcaagcat caccatcatt caggccatca
     2581 ccataccgga caccaccatc attcaggcag tcatcaccat accggatcgg actcagaagt
     2641 caatcaagaa gctaagccag aggtcaagcc agaagtcaag cctgagactc acatcaattt
     2701 aaaggtgtcc gatggatctt cagagatctt cttcaagatc aaaaagacca ctcctttaag
     2761 aaggctgatg gaagcgttcg ctaaaagaca gggtaaggaa atggactcct taagattctt
     2821 gtacgacggt attagaattc aagctgatca gacccctgaa gatttggaca tggaggataa
     2881 cgatattatt gaggctcaca gagaacagat tggtggcagc aaaatgtccg atgttaaatg
     2941 caccagcgtt gttctgctga gcgttctgca gcagctgcgt gttgaaagca gcagcaaact
     3001 gtgggcacag tgtgttcagc tgcataatga tattctgctg gcaaaagata ccaccgaagc
     3061 ctttgaaaaa atggttagcc tgctgagtgt gctgctgagc atgcagggtg cagttgatat
     3121 taacaaactg tgtgaagaga tgctggataa tcgtgcaacc ctgcagtaat aagcggccgc
     3181 acatcaccat cactaagctt aattagctga gcttggactc ctgttgatag atccagtaat
     3241 gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg cgttttttat
     3301 tggtgagaat ccaagctagc catgaaaata aactgtctgc ttacataaac agtaatacaa
     3361 ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca
     3421 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     3481 cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     3541 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     3601 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgct tggttactca
     3661 cgactgcgat ccccggcaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     3721 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     3781 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     3841 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     3901 tctggaaaga aatgcacaaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     3961 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     4021 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     4081 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     4141 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgggc
     4201 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
     4261 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     4321 tggcagggcg gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggggta
     4381 atgactctct agcttgaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     4441 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccctctag
     4501 attacgtgca gtcgatgata agctgtcaaa catgaga
//
