LOCUS       sequence-338013-        4273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SARS-COV-2 Nsp9 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4273)
  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 4273)
  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: pIA1414
FEATURES             Location/Qualifiers
     source          1..4273
                     /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             1800..1844
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(1882..1900)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1896..1943
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(2173..2988)
                     /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(2173..2988)
                     /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     2896..2915
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2989..3080)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3096..3845)
                     /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(3106..3123)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(3346..3365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4041..4273,1..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 gaaacaacga
     1441 actttctccg gtcgcgttgc gtcaaatgtc ttgcgctgct ggcaccaccc aaacggcgtg
     1501 tacggatgac aacgccttag catactataa tactacaaaa ggtggccgtt tcgtgcttgc
     1561 tttgttatct gatttacagg atttgaaatg ggcacgtttt cctaagagtg acggaacggg
     1621 aaccatttat acggaattag agcctccgtg ccgtttcgtc accgatacac caaaaggccc
     1681 gaaagtgaag tacctgtact tcatcaaggg tctgaataat ctgaatcgcg gcatggttct
     1741 gggtagtttg gctgcgaccg tgcgtctgca ataagcttgc ggccgcactc gagtctggta
     1801 aagaaaccgc tgctgcgaaa tttgaacgcc agcacatgga ctcgtctact agcgcagctt
     1861 aattaaccta ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     1921 aacgggtctt gaggggtttt ttgctgaaac ctcaggcatt tgagaagcac acggtcacac
     1981 tgcttccggt agtcaataaa ccggtaaacc agcaatagac ataagcggct atttaacgac
     2041 cctgccctga accgacgaca agctgacgac cgggtctccg caagtggcac ttttcgggga
     2101 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2161 atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc
     2221 aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc
     2281 gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac
     2341 atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc
     2401 atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg
     2461 ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt
     2521 cattcgtgat tgcgcctgag cgagacgaaa tacgcggtcg ctgttaaaag gacaattaca
     2581 aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc
     2641 tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg cagtggtgag
     2701 taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc
     2761 cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc
     2821 atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga ttgtcgcacc
     2881 tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga
     2941 atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcatac tcttcctttt
     3001 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3061 tatttagaaa aataaacaaa taggcatgca gcgctcttcc gcttcctcgc tcactgactc
     3121 gctacgctcg gtcgttcgac tgcggcgagc ggtgtcagct cactcaaaag cggtaatacg
     3181 gttatccaca gaatcagggg ataaagccgg aaagaacatg tgagcaaaaa gcaaagcacc
     3241 ggaagaagcc aacgccgcag gcgtttttcc ataggctccg cccccctgac gagcatcaca
     3301 aaaatcgacg ctcaagccag aggtggcgaa acccgacagg actataaaga taccaggcgt
     3361 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     3421 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgttggtatc
     3481 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3541 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3601 tatcgccact ggcagcagcc attggtaact gatttagagg actttgtctt gaagttatgc
     3661 acctgttaag gctaaactga aagaacagat tttggtgagt gcggtcctcc aacccactta
     3721 ccttggttca aagagttggt agctcagcga accttgagaa aaccaccgtt ggtagcggtg
     3781 gtttttcttt atttatgaga tgatgaatca atcggtctat caagtcaacg aacagctatt
     3841 ccgttactct agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc
     3901 atacgatata agttgtaatt ctcatgttag tcatgccccg cgcccaccgg aaggagctga
     3961 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     4021 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4081 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     4141 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     4201 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     4261 gtggttaacg gcg
//
