LOCUS       sequence-338042-        6730 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SARS-COV-2 Nsp12 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6730)
  AUTHORS   Wang B, Svetlov D, Artsimovitch I
  TITLE     NMPylation and de-NMPylation of SARS-CoV-2 nsp9 by the NiRAN domain.
  JOURNAL   Nucleic Acids Res. 2021 Aug 5. pii: 6342456. doi: 
            10.1093/nar/gkab677.
  PUBMED    34352100
REFERENCE   2  (bases 1 to 6730)
  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: "Nucleic 
            Acids Res. 2021 Aug 5. pii: 6342456. doi: 10.1093/nar/gkab677."
COMMENT     Sequence Label: pIA1400
FEATURES             Location/Qualifiers
     source          1..6730
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     812..831
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(851..928)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     974..993
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        974..992
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    993..1017
                     /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             1070..1099
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1109..1126
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1139..1432
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             4257..4301
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4339..4357)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4353..4400
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4630..5445)
                     /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(4630..5445)
                     /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     5353..5372
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5446..5537)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5553..6302)
                     /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(5563..5580)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5803..5822)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6498..6730,1..850))
                     /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 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa
       61 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
      121 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
      181 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
      241 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
      301 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
      361 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
      421 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
      481 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
      541 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
      601 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
      661 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
      721 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
      781 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
      841 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
      901 gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc
      961 tgcattagga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1021 gtagaaataa ttttgtttaa ctttaataag gagatatacc atgggcagcc accaccacca
     1081 ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccaca tggcaagcat
     1141 gtcggactca gaagtcaatc aagaagctaa gccagaggtc aagccagaag tcaagcctga
     1201 gactcacatc aatttaaagg tgtccgatgg atcttcagag atcttcttca agatcaaaaa
     1261 gaccactcct ttaagaaggc tgatggaagc gttcgctaaa agacagggta aggaaatgga
     1321 ctccttaaga ttcttgtacg acggtattag aatccaagct gatcagaccc ctgaagattt
     1381 ggacatggag gataacgata ttattgaggc tcacagagaa cagattggtg gatccgccga
     1441 cgcgcagtct tttctgaacc gcgtatgcgg ggtaagcgca gctcgcttga caccctgtgg
     1501 tactgggacg tctacagacg tggtttatcg cgcgttcgac atctataacg acaaagtcgc
     1561 cggtttcgct aagttcttga agaccaattg ctgtcgcttt caagagaaag atgaagatga
     1621 taatcttatt gactcttatt ttgtagtaaa gcgtcacacc ttcagcaact atcagcatga
     1681 agaaaccatc tataatcttt tgaaggactg tccagcagta gccaagcacg attttttcaa
     1741 atttcgtatt gacggtgaca tggtccccca catttcacgc cagcgcctta ccaaatacac
     1801 aatggcggat ctggtttatg ctcttcgtca ttttgacgaa ggaaattgcg acacccttaa
     1861 ggagattctg gtaacttata attgttgcga cgatgactat tttaataaaa aggattggta
     1921 tgattttgtc gaaaatccgg atattttacg tgtgtacgca aatctgggag agcgcgttcg
     1981 ccaagcgttg ttaaagacgg tacagttctg cgacgccatg cgtaatgccg gtatcgtggg
     2041 tgtgctgacc ttagataacc aagatcttaa cggtaattgg tatgatttcg gagattttat
     2101 tcagaccact cccggctccg gcgtgccggt agtggattcc tattatagtc tgctgatgcc
     2161 aatcttgacg ttgacccgtg ccttgacggc ggagagccat gtggacacag acttaacgaa
     2221 gccttacatt aaatgggatt tgcttaaata cgacttcacg gaggagcgtc ttaaactttt
     2281 tgaccgttac tttaagtatt gggaccagac gtatcacccc aactgcgtca actgcttaga
     2341 cgatcgctgc atcttacatt gtgcaaactt taatgtttta ttttccacag tttttccgcc
     2401 gacttccttc gggccactgg tacgcaagat ttttgtcgac ggcgtgccgt ttgtagtctc
     2461 tacggggtac cactttcgtg aacttggtgt tgttcataac caggatgtta atttacattc
     2521 gtcacgtctt tcattcaaag agttactggt atacgcggcc gatcccgcca tgcacgcagc
     2581 gtccgggaat ttacttctgg ataaacgtac tacgtgtttc agtgttgcag cgctgaccaa
     2641 caatgtagct ttccaaacag taaaaccagg gaatttcaat aaggattttt acgacttcgc
     2701 agtcagtaag ggttttttta aagaggggag ctcggttgag ttaaaacact tttttttcgc
     2761 tcaggatggg aacgctgcaa tctccgatta cgactactat cgctacaact tacctacgat
     2821 gtgcgatatt cgccaattac tgttcgtagt tgaagtagta gacaagtatt tcgactgtta
     2881 cgatggagga tgtattaatg cgaaccaagt aatcgtcaat aatttagata aatctgcagg
     2941 ttttcccttt aataagtggg gaaaggcgcg cctgtactac gattcaatgt cgtatgagga
     3001 ccaggacgct cttttcgcat ataccaaacg caatgttatc ccgacgatta cgcaaatgaa
     3061 tctgaaatat gccatttccg ctaaaaatcg cgctcgcacg gtcgcgggag tatcgatttg
     3121 ttctactatg actaaccgcc aattccatca aaagttgctt aaatctatcg cagcaacgcg
     3181 tggggcaacg gtggtcatcg ggacctcgaa gttttatgga ggttggcaca acatgctgaa
     3241 aaccgtgtat agtgacgtgg aaaacccgca tctgatgggg tgggattatc cgaaatgcga
     3301 tcgcgcaatg cctaatatgc ttcgcatcat ggcctcttta gtgctagcac gtaagcatac
     3361 gacatgttgc agcttgtccc atcgctttta ccgtttagct aatgagtgcg cacaagtatt
     3421 atcggagatg gtaatgtgcg gcgggagttt atacgtgaag ccaggtggta ctagttctgg
     3481 ggatgcaacc acagcgtatg ctaattcggt tttcaatatc tgtcaggccg ttaccgccaa
     3541 cgtaaacgca cttctgtcaa cggatggtaa taaaattgct gataagtacg tacgcaacct
     3601 gcaacatcgt ctgtatgagt gcctttatcg caaccgcgat gtcgacaccg actttgtaaa
     3661 cgagttctat gcatatttgc gtaagcattt ctctatgatg atcctttcag acgatgccgt
     3721 tgtgtgtttc aacagtacct atgcctcgca aggcctagta gcctccatca aaaactttaa
     3781 gtccgttctt tactatcaga acaacgtctt tatgtcagag gccaagtgct ggaccgagac
     3841 ggacttaact aaaggcccac acgagttctg ctcacaacat actatgcttg taaagcaagg
     3901 tgatgactac gtctatcttc cttatcctga cccaagccgc atcctgggag ctggttgttt
     3961 cgttgacgat atcgtgaaga ccgatggcac cctgatgatt gagcgtttcg tctctctggc
     4021 gattgacgcg tatccgttaa ctaagcaccc caatcaagaa tacgcggacg tttttcactt
     4081 atacttacag tatatccgta agttacacga tgagctgacc ggtcacatgc ttgatatgta
     4141 cagcgttatg ctgacgaatg acaacacttc gcgttattgg gagcctgagt tttatgaagc
     4201 gatgtataca cctcacacag ttctgcagta agcttgcggc cgcactcgag tctggtaaag
     4261 aaaccgctgc tgcgaaattt gaacgccagc acatggactc gtctactagc gcagcttaat
     4321 taacctaggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     4381 gggtcttgag gggttttttg ctgaaacctc aggcatttga gaagcacacg gtcacactgc
     4441 ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt taacgaccct
     4501 gccctgaacc gacgacaagc tgacgaccgg gtctccgcaa gtggcacttt tcggggaaat
     4561 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4621 aattaattct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     4681 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     4741 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     4801 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     4861 agtgacgact gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc
     4921 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     4981 tcgtgattgc gcctgagcga gacgaaatac gcggtcgctg ttaaaaggac aattacaaac
     5041 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     5101 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     5161 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     5221 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     5281 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     5341 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     5401 taatcgcggc ctagagcaag acgtttcccg ttgaatatgg ctcatactct tcctttttca
     5461 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     5521 ttagaaaaat aaacaaatag gcatgcagcg ctcttccgct tcctcgctca ctgactcgct
     5581 acgctcggtc gttcgactgc ggcgagcggt gtcagctcac tcaaaagcgg taatacggtt
     5641 atccacagaa tcaggggata aagccggaaa gaacatgtga gcaaaaagca aagcaccgga
     5701 agaagccaac gccgcaggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5761 atcgacgctc aagccagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5821 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5881 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt tggtatctca
     5941 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6001 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6061 cgccactggc agcagccatt ggtaactgat ttagaggact ttgtcttgaa gttatgcacc
     6121 tgttaaggct aaactgaaag aacagatttt ggtgagtgcg gtcctccaac ccacttacct
     6181 tggttcaaag agttggtagc tcagcgaacc ttgagaaaac caccgttggt agcggtggtt
     6241 tttctttatt tatgagatga tgaatcaatc ggtctatcaa gtcaacgaac agctattccg
     6301 ttactctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt cagccccata
     6361 cgatataagt tgtaattctc atgttagtca tgccccgcgc ccaccggaag gagctgactg
     6421 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     6481 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     6541 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     6601 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     6661 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     6721 gttaacggcg
//
