LOCUS       sequence-327702-        6721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Codon optimized SARS-CoV-2 Nsp12 gene was cloned into a modified 
            pRSFDuet-1 vector (Addgene #159107) for expression in E. coli..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6721)
  AUTHORS   Wang B, Svetlov V, Wolf YI, Koonin EV, Nudler E, Artsimovitch I
  TITLE     Allosteric Activation of SARS-CoV-2 RNA-Dependent RNA Polymerase by 
            Remdesivir Triphosphate and Other Phosphorylated Nucleotides.
  JOURNAL   mBio. 2021 Jun 29;12(3):e0142321. doi: 10.1128/mBio.01423-21. Epub 
            2021 Jun 22.
  PUBMED    34154407
REFERENCE   2  (bases 1 to 6721)
  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: "mBio."; 
            date: "2021-06-29"; volume: "12(3):e0142321. doi"; pages: " 
            10.1128/mBio.01423-21. Epub 2021 Jun 22"
COMMENT     Sequence Label: pIA1385
FEATURES             Location/Qualifiers
     source          1..6721
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     546..565
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(585..662)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     708..727
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        708..726
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    727..751
                     /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)."
     CDS             807..824
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             834..851
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             864..1157
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             3982..4026
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4064..4082)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4078..4125
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4355..5170)
                     /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(4355..5170)
                     /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     5078..5097
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5171..5262)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5278..6027)
                     /direction=LEFT
                     /label=RSF ori
                     /note="Plasmids containing the RSF 1030 origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     primer_bind     complement(5288..5305)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5528..5547)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6223..6721,1..584))
                     /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 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
       61 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
      121 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
      181 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
      241 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
      301 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
      361 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
      421 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
      481 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
      541 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
      601 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
      661 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaaattaa tacgactcac
      721 tataggggaa ttgtgagcgg ataacaattc ccctgtagaa ataattttgt ttaactttaa
      781 taaggagata taccatgggc agcagccatc atcatcatca tcacagcagc ggcctggtgc
      841 cgcgcggcag ccacatggca agcatgtcgg actcagaagt caatcaagaa gctaagccag
      901 aggtcaagcc agaagtcaag cctgagactc acatcaattt aaaggtgtcc gatggatctt
      961 cagagatctt cttcaagatc aaaaagacca ctcctttaag aaggctgatg gaagcgttcg
     1021 ctaaaagaca gggtaaggaa atggactcct taagattctt gtacgacggt attagaatcc
     1081 aagctgatca gacccctgaa gatttggaca tggaggataa cgatattatt gaggctcaca
     1141 gagaacagat tggtggatcc gccgacgcgc agtcttttct gaaccgcgta tgcggggtaa
     1201 gcgcagctcg cttgacaccc tgtggtactg ggacgtctac agacgtggtt tatcgcgcgt
     1261 tcgacatcta taacgacaaa gtcgccggtt tcgctaagtt cttgaagacc aattgctgtc
     1321 gctttcaaga gaaagatgaa gatgataatc ttattgactc ttattttgta gtaaagcgtc
     1381 acaccttcag caactatcag catgaagaaa ccatctataa tcttttgaag gactgtccag
     1441 cagtagccaa gcacgatttt ttcaaatttc gtattgacgg tgacatggtc ccccacattt
     1501 cacgccagcg ccttaccaaa tacacaatgg cggatctggt ttatgctctt cgtcattttg
     1561 acgaaggaaa ttgcgacacc cttaaggaga ttctggtaac ttataattgt tgcgacgatg
     1621 actattttaa taaaaaggat tggtatgatt ttgtcgaaaa tccggatatt ttacgtgtgt
     1681 acgcaaatct gggagagcgc gttcgccaag cgttgttaaa gacggtacag ttctgcgacg
     1741 ccatgcgtaa tgccggtatc gtgggtgtgc tgaccttaga taaccaagat cttaacggta
     1801 attggtatga tttcggagat tttattcaga ccactcccgg ctccggcgtg ccggtagtgg
     1861 attcctatta tagtctgctg atgccaatct tgacgttgac ccgtgccttg acggcggaga
     1921 gccatgtgga cacagactta acgaagcctt acattaaatg ggatttgctt aaatacgact
     1981 tcacggagga gcgtcttaaa ctttttgacc gttactttaa gtattgggac cagacgtatc
     2041 accccaactg cgtcaactgc ttagacgatc gctgcatctt acattgtgca aactttaatg
     2101 ttttattttc cacagttttt ccgccgactt ccttcgggcc actggtacgc aagatttttg
     2161 tcgacggcgt gccgtttgta gtctctacgg ggtaccactt tcgtgaactt ggtgttgttc
     2221 ataaccagga tgttaattta cattcgtcac gtctttcatt caaagagtta ctggtatacg
     2281 cggccgatcc cgccatgcac gcagcgtccg ggaatttact tctggataaa cgtactacgt
     2341 gtttcagtgt tgcagcgctg accaacaatg tagctttcca aacagtaaaa ccagggaatt
     2401 tcaataagga tttttacgac ttcgcagtca gtaagggttt ttttaaagag gggagctcgg
     2461 ttgagttaaa acactttttt ttcgctcagg atgggaacgc tgcaatctcc gattacgact
     2521 actatcgcta caacttacct acgatgtgcg atattcgcca attactgttc gtagttgaag
     2581 tagtagacaa gtatttcgac tgttacgatg gaggatgtat taatgcgaac caagtaatcg
     2641 tcaataattt agataaatct gcaggttttc cctttaataa gtggggaaag gcgcgcctgt
     2701 actacgattc aatgtcgtat gaggaccagg acgctctttt cgcatatacc aaacgcaatg
     2761 ttatcccgac gattacgcaa atgaatctga aatatgccat ttccgctaaa aatcgcgctc
     2821 gcacggtcgc gggagtatcg atttgttcta ctatgactaa ccgccaattc catcaaaagt
     2881 tgcttaaatc tatcgcagca acgcgtgggg caacggtggt catcgggacc tcgaagtttt
     2941 atggaggttg gcacaacatg ctgaaaaccg tgtatagtga cgtggaaaac ccgcatctga
     3001 tggggtggga ttatccgaaa tgcgatcgcg caatgcctaa tatgcttcgc atcatggcct
     3061 ctttagtgct agcacgtaag catacgacat gttgcagctt gtcccatcgc ttttaccgtt
     3121 tagctaatga gtgcgcacaa gtattatcgg agatggtaat gtgcggcggg agtttatacg
     3181 tgaagccagg tggtactagt tctggggatg caaccacagc gtatgctaat tcggttttca
     3241 atatctgtca ggccgttacc gccaacgtaa acgcacttct gtcaacggat ggtaataaaa
     3301 ttgctgataa gtacgtacgc aacctgcaac atcgtctgta tgagtgcctt tatcgcaacc
     3361 gcgatgtcga caccgacttt gtaaacgagt tctatgcata tttgcgtaag catttctcta
     3421 tgatgatcct ttcagacgat gccgttgtgt gtttcaacag tacctatgcc tcgcaaggcc
     3481 tagtagcctc catcaaaaac tttaagtccg ttctttacta tcagaacaac gtctttatgt
     3541 cagaggccaa gtgctggacc gagacggact taactaaagg cccacacgag ttctgctcac
     3601 aacatactat gcttgtaaag caaggtgatg actacgtcta tcttccttat cctgacccaa
     3661 gccgcatcct gggagctggt tgtttcgttg acgatatcgt gaagaccgat ggcaccctga
     3721 tgattgagcg tttcgtctct ctggcgattg acgcgtatcc gttaactaag caccccaatc
     3781 aagaatacgc ggacgttttt cacttatact tacagtatat ccgtaagtta cacgatgagc
     3841 tgaccggtca catgcttgat atgtacagcg ttatgctgac gaatgacaac acttcgcgtt
     3901 attgggagcc tgagttttat gaagcgatgt atacacctca cacagttctg cagtaagctt
     3961 gcggccgcac tcgagtctgg taaagaaacc gctgctgcga aatttgaacg ccagcacatg
     4021 gactcgtcta ctagcgcagc ttaattaacc taggctgctg ccaccgctga gcaataacta
     4081 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa acctcaggca
     4141 tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa ccagcaatag
     4201 acataagcgg ctatttaacg accctgccct gaaccgacga caagctgacg accgggtctc
     4261 cgcaagtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     4321 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     4381 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     4441 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     4501 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     4561 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     4621 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     4681 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcggt
     4741 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     4801 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     4861 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     4921 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     4981 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     5041 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     5101 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     5161 tatggctcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5221 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggcatg cagcgctctt
     5281 ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggtgtcag
     5341 ctcactcaaa agcggtaata cggttatcca cagaatcagg ggataaagcc ggaaagaaca
     5401 tgtgagcaaa aagcaaagca ccggaagaag ccaacgccgc aggcgttttt ccataggctc
     5461 cgcccccctg acgagcatca caaaaatcga cgctcaagcc agaggtggcg aaacccgaca
     5521 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5581 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5641 catagctcac gctgttggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5701 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5761 tccaacccgg taagacacga cttatcgcca ctggcagcag ccattggtaa ctgatttaga
     5821 ggactttgtc ttgaagttat gcacctgtta aggctaaact gaaagaacag attttggtga
     5881 gtgcggtcct ccaacccact taccttggtt caaagagttg gtagctcagc gaaccttgag
     5941 aaaaccaccg ttggtagcgg tggtttttct ttatttatga gatgatgaat caatcggtct
     6001 atcaagtcaa cgaacagcta ttccgttact ctagatttca gtgcaattta tctcttcaaa
     6061 tgtagcacct gaagtcagcc ccatacgata taagttgtaa ttctcatgtt agtcatgccc
     6121 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     6181 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     6241 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     6301 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     6361 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     6421 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     6481 atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact cggtaatggc
     6541 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     6601 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     6661 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     6721 a
//
