LOCUS       sequence-331060-        6785 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 6785)
  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 6785)
  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: nsp16_SARS-CoV-2|nsp10_SARS-CoV-2
FEATURES             Location/Qualifiers
     source          1..6785
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     26..45
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        129..147
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    148..172
                     /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             187..209
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             219..236
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             249..269
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        1243..1261
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1262..1286
                     /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             1778..1795
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1848..1866)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1862..1909
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1946..2401
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2033..2052)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2243..2264
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2493..3308)
                     /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(2493..3308)
                     /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     3216..3235
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3300..3319
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3430..4018
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3919..3938
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4172..4189
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4204..4343
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4269..4288)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4429..4451
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4445..4636)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5445..6527)
                     /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     6489..6508
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6528..6605)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
       61 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc
      121 cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga
      181 aataattttg tttaacttta agaaggagat ataccatgca tcatcatcat catcacagca
      241 gcggcagaga aaacttgtat ttccagggca gctcccaggc ttggcaacca ggagttgcca
      301 tgcccaactt gtacaaaatg caacgcatgc ttttggaaaa gtgtgacctg cagaattacg
      361 gtgacagcgc gactctgccc aaggggatca tgatgaatgt cgcaaagtat actcagttgt
      421 gccagtacct taatacgttg acgctggccg taccgtacaa catgcgcgta attcactttg
      481 gtgcgggcag cgacaaggga gtcgcgcctg gcacggctgt tttacgccaa tggttaccga
      541 ctggtacatt gctggttgac agtgacttga atgatttcgt aagcgatgcc gactcgacgc
      601 ttatcggcga ctgcgccacg gttcatacag ccaacaaatg ggacctgatt atttccgata
      661 tgtatgaccc aaaaaccaag aatgtgacaa aggagaacga ttcaaaggaa ggcttcttca
      721 cctatatctg tggttttatc cagcagaaat tagctcttgg cggttctgta gcaattaaga
      781 tcacggaaca ttcctggaac gcggatcttt acaagcttat gggccacttc gcgtggtgga
      841 ccgccttcgt gacgaacgtg aatgcaagtt cctctgaggc attcttgatc ggttgcaatt
      901 atcttggaaa acctcgtgag caaattgatg gctacgtgat gcacgccaac tatatcttct
      961 ggcgcaacac aaatcccatt cagttatcgt cctattcctt attcgatatg tctaagttcc
     1021 cgttgaagtt acgcggcact gccgtaatgt cgttgaagga agggcaaatt aatgatatga
     1081 ttttgtcgct tttatccaag gggcgtttaa tcatccgtga gaataatcgt gtggttatct
     1141 cgtctgacgt actggttaac aattgatgac gaagcttgcg gccgcataat gcttaagtcg
     1201 aacagaaagt aatcgtattg tacacggccg cataatcgaa attaatacga ctcactatag
     1261 gggaattgtg agcggataac aattccccat cttagtatat tagttaagta taagaaggag
     1321 atatacatat ggccggtaat gcaaccgaag tgcccgctaa cagcactgtt ctttccttct
     1381 gcgccttcgc ggtggatgcg gctaaagcct ataaagacta tcttgcttct gggggtcaac
     1441 ctattactaa ttgtgtcaaa atgctttgca cccacacagg gactggacag gctattaccg
     1501 tgacccctga ggctaatatg gatcaggaat cctttggagg cgcgtcctgt tgcctgtact
     1561 gccgttgtca tattgaccat cccaatccta aagggttttg cgacttaaaa ggaaagtatg
     1621 ttcaaattcc aacgacttgc gcaaatgacc ccgtgggatt cacattgaaa aacactgtat
     1681 gcactgtctg cgggatgtgg aaaggatacg ggtgttcatg cgatcaatta cgcgagccta
     1741 tgcttcagtg atgacgaagc ttgcggccgc actcgagcac caccaccacc accactgaga
     1801 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     1861 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     1921 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     1981 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2041 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2101 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2161 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2221 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2281 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2341 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     2401 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2461 attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga
     2521 aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt
     2581 aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct
     2641 gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg
     2701 ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta
     2761 tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc
     2821 gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg
     2881 ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc
     2941 gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc
     3001 ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg
     3061 gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca
     3121 ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac
     3181 aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat
     3241 aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata
     3301 tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac
     3361 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3421 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3481 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3541 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3601 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3661 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     3721 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     3781 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     3841 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     3901 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     3961 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     4021 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     4081 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     4141 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     4201 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc
     4261 actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc
     4321 tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac
     4381 gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
     4441 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat
     4501 cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga
     4561 gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt
     4621 tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga
     4681 taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt
     4741 tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa
     4801 tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc
     4861 agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt
     4921 ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg
     4981 ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag
     5041 taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc
     5101 gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac
     5161 cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga
     5221 tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca
     5281 cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc
     5341 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     5401 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     5461 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     5521 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     5581 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     5641 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     5701 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     5761 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     5821 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     5881 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     5941 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     6001 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     6061 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     6121 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     6181 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     6241 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     6301 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     6361 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     6421 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     6481 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     6541 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     6601 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     6661 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     6721 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     6781 cccga
//
