LOCUS       sequence-311604-        7323 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression plasmid for COVID-SARS2 NSP13 helicase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7323)
  AUTHORS   Newman JA, Douangamath A, Yadzani S, Yosaatmadja Y, Aimon A, 
            Brandao-Neto J, Dunnett L, Gorrie-Stone T, Skyner R, Fearon D, 
            Schapira M, von Delft F, Gileadi O
  TITLE     Structure, mechanism and crystallographic fragment screening of the 
            SARS-CoV-2 NSP13 helicase.
  JOURNAL   Nat Commun. 2021 Aug 11;12(1):4848. doi: 10.1038/s41467-021-25166-6.
  PUBMED    34381037
REFERENCE   2  (bases 1 to 7323)
  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: "2021-08-11"; volume: "12(1):4848. doi"; pages: " 
            10.1038/s41467-021-25166-6"
COMMENT     Sequence Label: COVID-SARS2 NSP13
FEATURES             Location/Qualifiers
     source          1..7323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1872..1889
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1942..1960)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1956..2003
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2040..2495
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2127..2146)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2337..2358
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2587..3402)
                     /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(2587..3402)
                     /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     3310..3329
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3394..3413
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3524..4112
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4013..4032
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4266..4283
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4298..4437
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4363..4382)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4523..4545
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4539..4730)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5539..6621)
                     /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     6583..6602
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6622..6699)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6905..6924
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7008..7027
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7008..7026
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7027..7051
                     /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             7066..7088
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7099..7116
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7126..7296
                     /codon_start=1
                     /product="Z domain of staphylococcal protein A, engineered 
                     to contain multiple positive charges (Hedhammar and Hober, 
                     2007)"
                     /label=Z-basic tag
                     /note="useful for purifying tagged proteins by 
                     cation-exchange chromatography"
                     /translation="VDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLL
                     AEAKKLNDAQPK"
     CDS             7303..7323
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
ORIGIN
        1 atggctgttg gtgcatgcgt tttgtgtaat agtcaaactt ctttgcgttg cggcgcctgc
       61 atccgccgcc catttctgtg ctgtaaatgt tgctacgatc atgtgattag cacgtcgcat
      121 aagctggtgc tgtcggtcaa cccgtatgtt tgtaatgcgc ccggttgtga cgtcacggat
      181 gttacccagc tctatctggg tggtatgtct tactactgca agtcacataa gccgccgatt
      241 tcatttcctc tctgcgcgaa tggccaggtg tttggtttgt ataaaaacac ctgcgtgggg
      301 tctgataatg taaccgattt taacgcgatt gctacgtgtg actggaccaa cgccggcgat
      361 tatatccttg ccaacacctg cactgaacgt cttaaactct tcgcggccga aaccctgaaa
      421 gcaaccgaag aaacgttcaa actgagctat ggtatcgcga cagtacgcga agtactcagt
      481 gaccgggagt tgcacctgtc gtgggaagtc ggtaaacctc gccccccgtt aaaccgcaac
      541 tacgtgttca ccggctatcg cgttaccaaa aacagcaagg tgcaaattgg tgagtacacc
      601 tttgagaaag gcgactatgg tgatgccgta gtataccgcg gcaccacgac ttacaaactg
      661 aatgtgggtg attattttgt attaacctca cataccgtaa tgccgttgag cgcccccacc
      721 ctcgtaccgc aggaacacta tgtacgtatc acgggtttat acccgacgct gaacatcagc
      781 gatgagttta gcagtaacgt tgctaactac cagaaagtgg gtatgcaaaa atactcgacg
      841 ttacagggcc cgccaggtac tggaaagtct catttcgcca tcggcttagc cttatactat
      901 ccatcagctc gcattgttta cacggcctgc tctcatgctg cagtggacgc cttatgcgag
      961 aaggcgttaa aatatctgcc gattgataaa tgctcccgta ttatcccggc gcgggcgcgg
     1021 gtcgagtgtt ttgacaaatt taaagtgaac agcacactgg aacagtatgt tttttgtacg
     1081 gtgaatgctc tcccagagac aactgccgat atcgtcgtgt tcgacgaaat cagcatggcc
     1141 actaactacg atctgtcggt cgttaacgcg cgtctccgcg ctaagcatta tgtttacatt
     1201 ggcgatccgg cccagctgcc ggctccacgc acgctcctga ctaaaggtac tctggaaccg
     1261 gaatatttta atagcgtatg ccgtctgatg aaaacgattg gacctgatat gttcctggga
     1321 acttgccgcc ggtgtccggc cgagatcgtt gatacggttt ctgctctggt ttatgacaat
     1381 aaattaaaag ctcacaaaga taaaagcgcg caatgcttca aaatgtttta taaaggtgta
     1441 attacccacg atgtgagtag cgcgattaac cgcccgcaaa ttggcgttgt ccgtgaattc
     1501 ctgacccgga atcctgcgtg gcgcaaagca gtgttcatta gcccatataa tagtcaaaac
     1561 gcggtagcgt ccaagatttt gggtctgccg acacaaaccg tggactccag ccagggcagt
     1621 gagtacgact atgtgatttt tacccagacc actgagacag cacactcctg caacgtgaac
     1681 cgcttcaatg tagcgatcac ccgtgcgaag gtaggtattt tgtgcatcat gtccgaccgt
     1741 gatctgtatg acaaattgca gtttaccagc ctggaaatcc ctcgccgtaa cgtggcaacc
     1801 cttcaataac agtaaaggtg gatacggatc cgaattcgag ctccgtcgac aagcttgcgg
     1861 ccgcactcga gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg
     1921 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     1981 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga
     2041 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2101 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2161 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2221 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2281 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     2341 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     2401 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     2461 cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg
     2521 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat
     2581 taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     2641 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     2701 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     2761 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     2821 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     2881 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     2941 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     3001 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     3061 aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca
     3121 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     3181 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     3241 cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg
     3301 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     3361 tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact
     3421 gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt
     3481 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3541 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3601 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3661 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3721 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3781 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3841 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3901 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3961 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4021 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4081 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4141 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4201 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     4261 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     4321 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     4381 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     4441 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     4501 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     4561 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     4621 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     4681 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     4741 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     4801 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     4861 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     4921 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     4981 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     5041 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     5101 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     5161 gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg
     5221 gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg
     5281 tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg
     5341 tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag
     5401 aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact
     5461 gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt
     5521 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     5581 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     5641 ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga
     5701 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     5761 ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat
     5821 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     5881 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     5941 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     6001 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     6061 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     6121 cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     6181 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     6241 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     6301 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     6361 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     6421 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     6481 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     6541 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     6601 gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca
     6661 tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct
     6721 tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg
     6781 ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg
     6841 ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc
     6901 catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg
     6961 ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa tacgactcac
     7021 tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa
     7081 gaaggagata tacatatgca ccatcatcat catcattctt ctggtgtgga taacaagttc
     7141 aacaaggagc gtcgaagagc tcgccgtgaa attcgccatc tgccgaacct gaaccgcgaa
     7201 cagcgtcgcg catttattcg cagcctgcgc gatgatccga gccagagcgc gaacctgctg
     7261 gcggaagcga agaagctgaa cgatgcgcag ccgaagggta ccgagaacct gtacttccaa
     7321 tcc
//
