LOCUS       sequence-330626-        5723 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the SARS-CoV-2 protein nsp10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5723)
  AUTHORS   Perveen S, Khalili Yazdi A, Devkota K, Li F, Ghiabi P, Hajian T, 
            Loppnau P, Bolotokova A, Vedadi M
  TITLE     A High-Throughput RNA Displacement Assay for Screening SARS-CoV-2 
            nsp10-nsp16 Complex toward Developing Therapeutics for COVID-19.
  JOURNAL   SLAS Discov. 2021 Jan 10:2472555220985040. doi: 
            10.1177/2472555220985040.
  PUBMED    33423577
REFERENCE   2  (bases 1 to 5723)
  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: "SLAS 
            Discov. 2021 Jan 10:2472555220985040. doi: 
            10.1177/2472555220985040."
COMMENT     Sequence Label: pET28-MHL: nsp10_SARS-CoV-2
FEATURES             Location/Qualifiers
     source          1..5723
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     60..79
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        60..78
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    79..103
                     /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             118..140
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             150..167
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             180..200
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             647..664
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(717..735)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      731..778
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      815..1270
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(902..921)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1112..1133
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1362..2177)
                     /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(1362..2177)
                     /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     2085..2104
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2169..2188
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2299..2887
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2788..2807
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3041..3058
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3073..3212
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3138..3157)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3298..3320
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3314..3505)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4314..5396)
                     /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     5358..5377
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5397..5474)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5680..5699
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
       61 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
      121 gtttaacttt aagaaggaga tataccatgc atcatcatca tcatcacagc agcggcagag
      181 aaaacttgta tttccagggc gccggtaatg caaccgaagt gcccgctaac agcactgttc
      241 tttccttctg cgccttcgcg gtggatgcgg ctaaagccta taaagactat cttgcttctg
      301 ggggtcaacc tattactaat tgtgtcaaaa tgctttgcac ccacacaggg actggacagg
      361 ctattaccgt gacccctgag gctaatatgg atcaggaatc ctttggaggc gcgtcctgtt
      421 gcctgtactg ccgttgtcat attgaccatc ccaatcctaa agggttttgc gacttaaaag
      481 gaaagtatgt tcaaattcca acgacttgcg caaatgaccc cgtgggattc acattgaaaa
      541 acactgtatg cactgtctgc gggatgtgga aaggatacgg gtgttcatgc gatcaattac
      601 gcgagcctat gcttcagtga tgacgaagct tgcggccgca ctcgagcacc accaccacca
      661 ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
      721 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
      781 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
      841 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
      901 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
      961 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1021 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     1081 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     1141 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     1201 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     1261 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1321 tctaaataca ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc
     1381 atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc
     1441 cgtttctgta atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg
     1501 tatcggtctg cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca
     1561 aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc
     1621 aaaagtttat gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca
     1681 aaatcactcg catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat
     1741 acgcgatcgc tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac
     1801 actgccagcg catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat
     1861 gctgttttcc cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa
     1921 tgcttgatgg tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct
     1981 gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc
     2041 ttcccataca atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta
     2101 tacccatata aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc
     2161 cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt
     2221 gttcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     2281 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2341 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2401 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     2461 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2521 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2581 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2641 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     2701 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     2761 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     2821 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     2881 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     2941 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     3001 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     3061 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3121 caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc
     3181 cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg
     3241 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     3301 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt
     3361 aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca
     3421 gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa
     3481 gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg
     3541 gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac
     3601 atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc
     3661 agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac
     3721 agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact
     3781 tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg
     3841 tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct
     3901 gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca
     3961 tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg
     4021 tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg
     4081 acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg
     4141 ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga
     4201 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     4261 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     4321 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4381 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     4441 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     4501 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     4561 ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac
     4621 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     4681 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     4741 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     4801 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     4861 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     4921 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     4981 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     5041 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     5101 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     5161 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     5221 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     5281 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     5341 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     5401 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     5461 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca
     5521 gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga
     5581 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
     5641 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
     5701 gccagcaacc gcacctgtgg cgc
//
