LOCUS       sequence-330624-        6200 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of the SARS-CoV-2 protein nsp16.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6200)
  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 6200)
  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
FEATURES             Location/Qualifiers
     source          1..6200
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     34..53
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        34..52
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    53..77
                     /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             92..114
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             124..141
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             154..174
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1098..1115
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1168..1186)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1182..1229
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1266..1721
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1353..1372)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1563..1584
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1813..2628)
                     /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(1813..2628)
                     /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     2536..2555
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2620..2639
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2750..3338
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3239..3258
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3492..3509
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3524..3663
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3589..3608)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3749..3771
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3765..3956)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4765..5847)
                     /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     5809..5828
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5848..5925)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6131..6150
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
       61 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc
      121 atgcatcatc atcatcatca cagcagcggc agagaaaact tgtatttcca gggcagctcc
      181 caggcttggc aaccaggagt tgccatgccc aacttgtaca aaatgcaacg catgcttttg
      241 gaaaagtgtg acctgcagaa ttacggtgac agcgcgactc tgcccaaggg gatcatgatg
      301 aatgtcgcaa agtatactca gttgtgccag taccttaata cgttgacgct ggccgtaccg
      361 tacaacatgc gcgtaattca ctttggtgcg ggcagcgaca agggagtcgc gcctggcacg
      421 gctgttttac gccaatggtt accgactggt acattgctgg ttgacagtga cttgaatgat
      481 ttcgtaagcg atgccgactc gacgcttatc ggcgactgcg ccacggttca tacagccaac
      541 aaatgggacc tgattatttc cgatatgtat gacccaaaaa ccaagaatgt gacaaaggag
      601 aacgattcaa aggaaggctt cttcacctat atctgtggtt ttatccagca gaaattagct
      661 cttggcggtt ctgtagcaat taagatcacg gaacattcct ggaacgcgga tctttacaag
      721 cttatgggcc acttcgcgtg gtggaccgcc ttcgtgacga acgtgaatgc aagttcctct
      781 gaggcattct tgatcggttg caattatctt ggaaaacctc gtgagcaaat tgatggctac
      841 gtgatgcacg ccaactatat cttctggcgc aacacaaatc ccattcagtt atcgtcctat
      901 tccttattcg atatgtctaa gttcccgttg aagttacgcg gcactgccgt aatgtcgttg
      961 aaggaagggc aaattaatga tatgattttg tcgcttttat ccaaggggcg tttaatcatc
     1021 cgtgagaata atcgtgtggt tatctcgtct gacgtactgg ttaacaattg atgacgaagc
     1081 ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct aacaaagccc
     1141 gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg
     1201 cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc ggattggcga
     1261 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1321 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1381 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1441 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1501 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     1561 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     1621 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     1681 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     1741 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1801 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     1861 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     1921 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     1981 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     2041 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     2101 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     2161 cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca
     2221 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     2281 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     2341 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     2401 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     2461 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     2521 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     2581 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa caccccttgt
     2641 attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct taacgtgagt
     2701 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     2761 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     2821 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     2881 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     2941 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3001 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3061 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3121 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3181 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3241 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3301 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3361 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3421 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3481 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3541 tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta caatctgctc
     3601 tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg ggtcatggct
     3661 gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca
     3721 tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg
     3781 tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat
     3841 tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag aagcgttaat
     3901 gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt ggtcactgat
     3961 gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa acgagagagg
     4021 atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg ttgtgagggt
     4081 aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg tcaatgccag
     4141 cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc tgcgatgcag
     4201 atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta cgaaacacgg
     4261 aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt
     4321 cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc ccgccagcct
     4381 agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc catgccggcg
     4441 ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg
     4501 agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga
     4561 aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg
     4621 ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag
     4681 ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc
     4741 taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4801 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag
     4861 ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc
     4921 ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt
     4981 gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac
     5041 cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc
     5101 catctgatcg ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat
     5161 ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat
     5221 ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact
     5281 taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc
     5341 cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct ggtcagagac
     5401 atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc
     5461 atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc
     5521 cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag
     5581 ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact
     5641 ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt
     5701 gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac
     5761 gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc
     5821 gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct cttccgggcg
     5881 ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct
     5941 ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg ccgttgagca
     6001 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
     6061 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
     6121 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
     6181 tgccggccac gatgcgtccg
//
