LOCUS       sequence-307616-        6340 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of Sars-CoV2 Nsp9 protein with His-tag and 
            GST-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6340)
  AUTHORS   Mihalic F, Benz C, Kassa E, Lindqvist R, Simonetti L, Inturi R, 
            Aronsson H, Andersson E, Chi CN, Davey NE, Overby AK, Jemth P, 
            Ivarsson Y
  TITLE     Identification of motif-based interactions between SARS-CoV-2 
            protein domains and human peptide ligands pinpoint antiviral 
            targets.
  JOURNAL   Nat Commun. 2023 Sep 13;14(1):5636. doi: 10.1038/s41467-023-41312-8.
  PUBMED    37704626
REFERENCE   2  (bases 1 to 6340)
  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: "Nat 
            Commun."; date: "2023-09-13"; volume: "14(1):5636. doi"; pages: " 
            10.1038/s41467-023-41312-8"
COMMENT     Sequence Label: pETM33_Nsp9
FEATURES             Location/Qualifiers
     source          1..6340
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     52..71
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(91..168)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     374..393
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     477..496
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        477..495
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    496..520
                     /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             535..557
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             570..587
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             606..1259
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             1266..1289
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             1681..1698
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1751..1769)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1765..1812
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1849..2304
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1936..1955)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2146..2167
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2396..3211)
                     /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(2396..3211)
                     /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     3119..3138
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3203..3222
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3333..3921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3822..3841
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4075..4092
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4107..4246
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4172..4191)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4332..4354
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4348..4539)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5348..6340,1..90))
                     /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"
ORIGIN
        1 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
       61 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
      121 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
      181 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
      241 gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca
      301 cggggcctgc caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc
      361 gatcttcccc atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg
      421 tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat
      481 acgactcact ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt
      541 taactttaag aaggagatat accatgaaac atcaccatca ccatcacaac actagtagca
      601 attccatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac
      661 ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg
      721 ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata
      781 ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc
      841 acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg
      901 ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca
      961 aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc
     1021 ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc
     1081 ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt
     1141 ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata
     1201 tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat
     1261 cggatctgga agttctgttc caggggccca tggacggtaa caacgagctg agcccggttg
     1321 cgctgcgtca gatgagctgc gcggcgggca ccacccaaac cgcgtgcacc gacgataacg
     1381 cgctggcgta ctataacacc accaagggtg gccgtttcgt tctggcgctg ctgagcgacc
     1441 tgcaggatct gaagtgggcg cgttttccga aaagcgacgg caccggcacc atctacaccg
     1501 agctggaacc gccgtgccgt ttcgtgaccg ataccccgaa gggtccgaag gttaagtacc
     1561 tgtacttcat caagggcctg aacaacctga accgtggtat ggtgctgggc agcctggcgg
     1621 cgaccgttcg tctgcaataa gaattcgagc tccgtcgaca agcttgcggc cgcactcgag
     1681 caccaccacc accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg
     1741 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1801 aggggttttt tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta
     1861 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1921 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1981 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     2041 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     2101 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     2161 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     2221 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     2281 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2341 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga
     2401 aaaactcatc gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat
     2461 atttttgaaa aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga
     2521 tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta
     2581 atttcccctc gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat
     2641 ccggtgagaa tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat
     2701 tacgctcgtc atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct
     2761 gagcgagacg aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca
     2821 accggcgcag gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt
     2881 ctaatacctg gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag
     2941 gagtacggat aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc
     3001 tgaccatctc atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact
     3061 ctggcgcatc gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat
     3121 cgcgagccca tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag
     3181 agcaagacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     3241 cagacagttt tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3301 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3361 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3421 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     3481 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3541 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3601 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3661 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3721 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3781 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3841 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3901 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3961 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4021 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4081 tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc
     4141 ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     4201 gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc
     4261 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4321 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4381 gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg
     4441 ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa
     4501 gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg
     4561 atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt
     4621 tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg
     4681 gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga
     4741 tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt
     4801 gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca
     4861 tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat
     4921 cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga
     4981 caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc
     5041 gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc
     5101 gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa
     5161 aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat
     5221 aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc
     5281 tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc
     5341 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5401 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     5461 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     5521 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     5581 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     5641 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     5701 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     5761 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     5821 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     5881 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     5941 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     6001 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     6061 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     6121 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     6181 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     6241 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     6301 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
//
