LOCUS       sequence-307733-        7042 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression of Sars-CoV2 Nsp15 protein with His-tag and 
            GST-tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7042)
  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 7042)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_Nsp15
FEATURES             Location/Qualifiers
     source          1..7042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      43..631
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     532..551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     785..802
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    817..956
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(882..901)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1042..1064
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1058..1249)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2058..3140)
                     /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     3102..3121
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3141..3218)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3424..3443
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3527..3546
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3527..3545
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3546..3570
                     /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             3585..3607
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3620..3637
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3656..4309
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             4316..4339
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             5433..5450
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5503..5521)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5517..5564
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5601..6056
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5688..5707)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5898..5919
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(6148..6963)
                     /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(6148..6963)
                     /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     6871..6890
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6955..6974
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
       61 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
      121 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
      181 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
      241 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
      301 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
      361 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
      421 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
      481 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
      541 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
      601 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
      661 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
      721 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
      781 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
      841 gcatctgtgc ggtatttcac accgcaatgg tgcactctca gtacaatctg ctctgatgcc
      901 gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc
      961 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     1021 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     1081 cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga
     1141 tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc
     1201 ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg
     1261 tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca
     1321 cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac
     1381 tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg
     1441 ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga
     1501 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
     1561 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
     1621 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
     1681 tcctcaacga caggagcacg atcatgcgca cccgtggggc cgccatgccg gcgataatgg
     1741 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     1801 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     1861 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     1921 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     1981 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     2041 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     2101 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     2161 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     2221 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     2281 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     2341 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     2401 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     2461 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     2521 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     2581 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     2641 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     2701 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     2761 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     2821 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     2881 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     2941 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     3001 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     3061 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     3121 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     3181 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     3241 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     3301 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     3361 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     3421 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     3481 cacgatgcgt ccggcgtaga ggatcgagat ctcgatcccg cgaaattaat acgactcact
     3541 ataggggaat tgtgagcgga taacaattcc cctctagaaa taattttgtt taactttaag
     3601 aaggagatat accatgaaac atcaccatca ccatcacaac actagtagca attccatgtc
     3661 ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga
     3721 atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg
     3781 aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga
     3841 tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt
     3901 gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat
     3961 tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt
     4021 tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata
     4081 tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt
     4141 tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg
     4201 tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc
     4261 tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat cggatctgga
     4321 agttctgttc caggggccca tggacggtct gcagagcctg gagaacgttg cgtttaacgt
     4381 ggttaacaag ggccacttcg acggtcagca aggcgaagtg ccggttagca tcattaacaa
     4441 caccgtgtac accaaggttg acggtgtgga tgttgagctg tttgaaaaca aaaccaccct
     4501 gccggtgaac gttgcgttcg agctgtgggc gaagcgtaac atcaaaccgg tgccggaagt
     4561 taagattctg aacaacctgg gtgtggacat cgcggcgaac accgttattt gggactataa
     4621 acgtgatgcg ccggcgcaca tcagcaccat tggcgtttgc agcatgaccg acatcgcgaa
     4681 gaaaccgacc gaaaccatct gcgcgccgct gaccgtgttc tttgacggtc gtgtggatgg
     4741 ccaggttgac ctgtttcgta acgcgcgtaa cggtgtgctg atcaccgagg gtagcgttaa
     4801 aggcctgcag ccgagcgtgg gtccgaaaca agcgagcctg aacggtgtta ccctgattgg
     4861 cgaagcggtg aagacccagt tcaactacta taagaaagtt gacggcgtgg ttcagcaact
     4921 gccggaaacc tactttaccc agagccgtaa cctgcaagag ttcaagccgc gtagccaaat
     4981 ggagatcgat tttctggaac tggcgatgga cgagttcatt gaacgttaca aactggaggg
     5041 ttatgcgttt gaacacatcg tttacggcga tttcagccac agccaactgg gtggcctgca
     5101 cctgctgatt ggtctggcga agcgtttcaa agagagcccg tttgagctgg aagatttcat
     5161 cccgatggac agcaccgtga agaactattt tattaccgat gcgcagaccg gcagcagcaa
     5221 atgcgtgtgc agcgttatcg acctgctgct ggacgatttc gttgaaatca ttaaaagcca
     5281 ggacctgagc gtggtgagca aggtggttaa agtgaccatc gattacaccg agattagctt
     5341 tatgctgtgg tgcaaggacg gtcacgtgga aaccttctat ccgaaactgt aagaattcga
     5401 gctccgtcga caagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg
     5461 ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag
     5521 cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta
     5581 tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     5641 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     5701 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     5761 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     5821 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5881 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     5941 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     6001 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt
     6061 ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca
     6121 aatatgtatc cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg
     6181 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     6241 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     6301 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     6361 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     6421 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     6481 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     6541 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     6601 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg ttttcccggg
     6661 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     6721 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     6781 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaatcg
     6841 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     6901 agcatccatg ttggaattta atcgcggcct agagcaagac gtttcccgtt gaatatggct
     6961 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgaccaaaa
     7021 tcccttaacg tgagttttcg tt
//
