LOCUS       sequence-310625-        7104 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the SARS-CoV-2 nsp13 protein with an N-terminal His6 tag 
            followed by an HRV 3C/PreScission cleavage site..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7104)
  AUTHORS   Chen J, Malone B, Llewellyn E, Grasso M, Shelton PMM, Olinares PDB, 
            Maruthi K, Eng ET, Vatandaslar H, Chait BT, Kapoor TM, Darst SA, 
            Campbell EA
  TITLE     Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 
            Replication-Transcription Complex.
  JOURNAL   Cell. 2020 Sep 17;182(6):1560-1573.e13. doi: 
            10.1016/j.cell.2020.07.033. Epub 2020 Jul 28.
  PUBMED    32783916
REFERENCE   2  (bases 1 to 7104)
  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"; doi: 
            "10.1016/j.cell.2020.07.033"; journalName: "Cell"; date: 
            "2020-09-17- 17"; volume: "182"; issue: "6"; pages: "1560-1573.e13"
COMMENT     Sequence Label: pET28a 6xHis PreScission SARS-CoV-2 nsp13
FEATURES             Location/Qualifiers
     source          1..7104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     434..453
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(473..550)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     756..775
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     859..878
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        859..877
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    878..902
                     /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             917..939
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             958..975
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             985..1008
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2827..2844
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2897..2915)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2911..2958
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2995..3450
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3082..3101)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3292..3313
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3542..4357)
                     /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(3542..4357)
                     /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     4265..4284
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4349..4368
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4479..5067
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4968..4987
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5221..5238
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5253..5392
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5318..5337)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5478..5500
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5494..5685)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6494..7104,1..472))
                     /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 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
       61 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
      121 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
      181 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
      241 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
      301 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
      361 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
      421 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
      481 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt
      541 cgatggtgtc cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc
      601 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
      661 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
      721 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
      781 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag
      841 atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg gataacaatt
      901 cccctctaga aataattttg tttaacttta agaaggagat ataccatggg cagcagccat
      961 catcatcatc atcacagcag cggcctggag gtgctgttcc agggacctca tatggctgtg
     1021 ggtgcctgcg tcctgtgcaa ctctcagact tctctgaggt gcggtgcttg catcaggcgt
     1081 cccttcctgt gctgcaagtg ctgctacgac cacgtcatct ctacttcaca caagctggtg
     1141 ctgtccgtca acccatacgt gtgcaacgct cctggttgcg acgtgactga cgtcacccag
     1201 ctgtacctgg gtggcatgtc ctactactgc aagagccaca agcctcccat cagcttccca
     1261 ctgtgcgcca acggtcaggt gttcggcctg tacaagaaca cctgcgtggg ttctgacaac
     1321 gtcactgact tcaacgctat cgccacctgc gactggacta acgctggcga ctacatcctg
     1381 gccaacactt gcaccgaacg tctgaagctg ttcgctgccg agaccctgaa ggctactgag
     1441 gaaaccttca agctgtcata cggaatcgcc actgtgagag aggtcctgtc tgaccgcgaa
     1501 ctgcacctgt catgggaggt cggcaagcct aggccacctc tgaacagaaa ctacgtgttc
     1561 accggctacc gtgtcactaa gaactccaag gtgcagatcg gagagtacac cttcgaaaag
     1621 ggcgactacg gagacgctgt ggtctacagg ggcaccacta cctacaagct gaacgtggga
     1681 gactacttcg tcctgacttc ccacaccgtg atgccactga gcgcccctac tctggtcccc
     1741 caggaacact acgtgcgcat caccggcctg tacccaactc tgaacatcag cgacgagttc
     1801 tccagcaacg tcgctaacta ccagaaagtg ggtatgcaga agtactctac cctgcagggt
     1861 cctcctggaa ctggcaagtc acacttcgct atcggactgg ccctgtacta cccttccgct
     1921 agaatcgtct acactgcttg ctctcacgct gctgtggacg ctctgtgcga aaaggccctg
     1981 aagtacctgc ccatcgacaa gtgctcacgc atcatcccag ctagggccag agtcgaatgc
     2041 ttcgacaagt tcaaggtgaa ctccaccctg gagcagtacg tgttctgcac tgtcaacgct
     2101 ctgcccgaaa ctaccgccga catcgtggtc ttcgacgaga tctctatggc taccaactac
     2161 gacctgtcag tggtcaacgc tcgcctgcgt gccaagcact acgtctacat cggagaccct
     2221 gctcagctgc ctgcccccag aactctgctg accaagggta ctctggagcc agaatacttc
     2281 aactctgtgt gccgcctgat gaagaccatc ggaccagaca tgttcctggg tacttgcagg
     2341 agatgccctg ctgaaatcgt ggacaccgtc tctgccctgg tgtacgacaa caagctgaag
     2401 gctcacaagg acaagtcagc ccagtgcttc aagatgttct acaagggcgt gatcacccac
     2461 gacgtctctt cagccatcaa ccgtcctcag atcggagtgg tccgcgagtt cctgactcgt
     2521 aaccctgctt ggaggaaggc cgtcttcatc tctccctaca actcacagaa cgctgtggcc
     2581 tccaagatcc tgggactgcc cactcagacc gtcgactcca gccagggtag cgaatacgac
     2641 tacgtgatct tcacccagac taccgagact gctcactctt gcaacgtgaa ccgtttcaac
     2701 gtcgctatca ccagggccaa ggtcggcatc ctgtgcatca tgtccgacag ggacctgtac
     2761 gacaagctgc agttcaccag cctggagatc cctcgccgta acgtggccac tctgcagtaa
     2821 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
     2881 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     2941 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     3001 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3061 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3121 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3181 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3241 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3301 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3361 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3421 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     3481 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     3541 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     3601 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     3661 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     3721 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     3781 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     3841 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     3901 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     3961 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     4021 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     4081 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     4141 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     4201 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     4261 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     4321 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     4381 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     4441 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4501 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     4561 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     4621 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     4681 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     4741 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4801 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4861 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4921 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4981 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     5041 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     5101 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     5161 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     5221 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     5281 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     5341 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     5401 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     5461 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     5521 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     5581 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     5641 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     5701 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     5761 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     5821 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     5881 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     5941 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     6001 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     6061 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     6121 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     6181 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     6241 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     6301 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     6361 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     6421 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     6481 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6541 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     6601 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     6661 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     6721 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     6781 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     6841 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     6901 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     6961 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     7021 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     7081 cgtcttcatg ggagaaaata atac
//
