LOCUS       sequence-331075-        6860 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the SARS-CoV-2 proteins nsp10 and nsp16 from
            a bicistronic mRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6860)
  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 6860)
  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: p28BIOHTEV-LIC: nsp16_SARS-CoV-2|nsp10_SARS-CoV-2
FEATURES             Location/Qualifiers
     source          1..6860
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        91..109
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    110..134
                     /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             149..171
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             184..228
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             250..270
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        1244..1262
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1263..1287
                     /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             1768..1785
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1815..1832
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1885..1903)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1899..1946
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1983..2438
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2070..2089)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2280..2301
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2530..3345)
                     /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(2530..3345)
                     /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     3253..3272
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3337..3356
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3467..4055
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3956..3975
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4209..4226
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4241..4380
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4306..4325)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4466..4488
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4482..4673)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5482..6564)
                     /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     6526..6545
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6565..6642)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     join(6848..6860,1..7)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
       61 tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag
      121 cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag atataccatg
      181 agcggcctga acgatatttt tgaagcgcag aaaattgaat ggcatgaagg cagcgctgga
      241 ggttcaggtg aaaacttgta tttccagggc agctcccagg cttggcaacc aggagttgcc
      301 atgcccaact tgtacaaaat gcaacgcatg cttttggaaa agtgtgacct gcagaattac
      361 ggtgacagcg cgactctgcc caaggggatc atgatgaatg tcgcaaagta tactcagttg
      421 tgccagtacc ttaatacgtt gacgctggcc gtaccgtaca acatgcgcgt aattcacttt
      481 ggtgcgggca gcgacaaggg agtcgcgcct ggcacggctg ttttacgcca atggttaccg
      541 actggtacat tgctggttga cagtgacttg aatgatttcg taagcgatgc cgactcgacg
      601 cttatcggcg actgcgccac ggttcataca gccaacaaat gggacctgat tatttccgat
      661 atgtatgacc caaaaaccaa gaatgtgaca aaggagaacg attcaaagga aggcttcttc
      721 acctatatct gtggttttat ccagcagaaa ttagctcttg gcggttctgt agcaattaag
      781 atcacggaac attcctggaa cgcggatctt tacaagctta tgggccactt cgcgtggtgg
      841 accgccttcg tgacgaacgt gaatgcaagt tcctctgagg cattcttgat cggttgcaat
      901 tatcttggaa aacctcgtga gcaaattgat ggctacgtga tgcacgccaa ctatatcttc
      961 tggcgcaaca caaatcccat tcagttatcg tcctattcct tattcgatat gtctaagttc
     1021 ccgttgaagt tacgcggcac tgccgtaatg tcgttgaagg aagggcaaat taatgatatg
     1081 attttgtcgc ttttatccaa ggggcgttta atcatccgtg agaataatcg tgtggttatc
     1141 tcgtctgacg tactggttaa caattgatga cgaagcttgc ggccgcataa tgcttaagtc
     1201 gaacagaaag taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata
     1261 ggggaattgt gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga
     1321 gatatacata tggccggtaa tgcaaccgaa gtgcccgcta acagcactgt tctttccttc
     1381 tgcgccttcg cggtggatgc ggctaaagcc tataaagact atcttgcttc tgggggtcaa
     1441 cctattacta attgtgtcaa aatgctttgc acccacacag ggactggaca ggctattacc
     1501 gtgacccctg aggctaatat ggatcaggaa tcctttggag gcgcgtcctg ttgcctgtac
     1561 tgccgttgtc atattgacca tcccaatcct aaagggtttt gcgacttaaa aggaaagtat
     1621 gttcaaattc caacgacttg cgcaaatgac cccgtgggat tcacattgaa aaacactgta
     1681 tgcactgtct gcgggatgtg gaaaggatac gggtgttcat gcgatcaatt acgcgagcct
     1741 atgcttcagt gatgaggtgg aagtggtcat caccaccatc atcactgatg acgaagcttg
     1801 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     1861 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     1921 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg
     1981 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     2041 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     2101 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     2161 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     2221 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     2281 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     2341 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     2401 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     2461 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2521 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     2581 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     2641 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     2701 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     2761 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     2821 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     2881 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     2941 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     3001 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     3061 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     3121 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     3181 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     3241 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     3301 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     3361 actgtttatg taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt
     3421 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3481 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3541 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3601 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3661 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3721 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3781 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3841 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3901 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3961 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     4021 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     4081 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     4141 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
     4201 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
     4261 ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga
     4321 tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg
     4381 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
     4441 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
     4501 tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca
     4561 cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc
     4621 tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc
     4681 tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg
     4741 ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa
     4801 caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc
     4861 ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc
     4921 cggaacataa tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa
     4981 ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac
     5041 gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc
     5101 cgggtcctca acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata
     5161 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     5221 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     5281 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     5341 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     5401 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     5461 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     5521 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     5581 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     5641 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     5701 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     5761 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     5821 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     5881 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     5941 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     6001 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     6061 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     6121 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     6181 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     6241 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     6301 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     6361 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     6421 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     6481 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     6541 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     6601 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     6661 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     6721 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6781 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6841 ccccatcggt gatgtcggcg
//
