LOCUS       sequence-338000-        6721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SARS-COV-2 Nsp12 D760A in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6721)
  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 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: "Nucleic 
            Acids Res. 2021 Aug 5. pii: 6342456. doi: 10.1093/nar/gkab677."
COMMENT     Sequence Label: pIA1408
FEATURES             Location/Qualifiers
     source          1..6721
                     /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             1073..1090
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1100..1117
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1130..1423
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             4248..4292
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4330..4348)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4344..4391
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4621..5436)
                     /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(4621..5436)
                     /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     5344..5363
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(5437..5528)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5544..6293)
                     /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(5554..5571)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(5794..5813)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6489..6721,1..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 atgggcagca gccatcatca
     1081 tcatcatcac agcagcggcc tggtgccgcg cggcagccac atggcaagca tgtcggactc
     1141 agaagtcaat caagaagcta agccagaggt caagccagaa gtcaagcctg agactcacat
     1201 caatttaaag gtgtccgatg gatcttcaga gatcttcttc aagatcaaaa agaccactcc
     1261 tttaagaagg ctgatggaag cgttcgctaa aagacagggt aaggaaatgg actccttaag
     1321 attcttgtac gacggtatta gaatccaagc tgatcagacc cctgaagatt tggacatgga
     1381 ggataacgat attattgagg ctcacagaga acagattggt ggatccgccg acgcgcagtc
     1441 ttttctgaac cgcgtatgcg gggtaagcgc agctcgcttg acaccctgtg gtactgggac
     1501 gtctacagac gtggtttatc gcgcgttcga catctataac gacaaagtcg ccggtttcgc
     1561 taagttcttg aagaccaatt gctgtcgctt tcaagagaaa gatgaagatg ataatcttat
     1621 tgactcttat tttgtagtaa agcgtcacac cttcagcaac tatcagcatg aagaaaccat
     1681 ctataatctt ttgaaggact gtccagcagt agccaagcac gattttttca aatttcgtat
     1741 tgacggtgac atggtccccc acatttcacg ccagcgcctt accaaataca caatggcgga
     1801 tctggtttat gctcttcgtc attttgacga aggaaattgc gacaccctta aggagattct
     1861 ggtaacttat aattgttgcg acgatgacta ttttaataaa aaggattggt atgattttgt
     1921 cgaaaatccg gatattttac gtgtgtacgc aaatctggga gagcgcgttc gccaagcgtt
     1981 gttaaagacg gtacagttct gcgacgccat gcgtaatgcc ggtatcgtgg gtgtgctgac
     2041 cttagataac caagatctta acggtaattg gtatgatttc ggagatttta ttcagaccac
     2101 tcccggctcc ggcgtgccgg tagtggattc ctattatagt ctgctgatgc caatcttgac
     2161 gttgacccgt gccttgacgg cggagagcca tgtggacaca gacttaacga agccttacat
     2221 taaatgggat ttgcttaaat acgacttcac ggaggagcgt cttaaacttt ttgaccgtta
     2281 ctttaagtat tgggaccaga cgtatcaccc caactgcgtc aactgcttag acgatcgctg
     2341 catcttacat tgtgcaaact ttaatgtttt attttccaca gtttttccgc cgacttcctt
     2401 cgggccactg gtacgcaaga tttttgtcga cggcgtgccg tttgtagtct ctacggggta
     2461 ccactttcgt gaacttggtg ttgttcataa ccaggatgtt aatttacatt cgtcacgtct
     2521 ttcattcaaa gagttactgg tatacgcggc cgatcccgcc atgcacgcag cgtccgggaa
     2581 tttacttctg gataaacgta ctacgtgttt cagtgttgca gcgctgacca acaatgtagc
     2641 tttccaaaca gtaaaaccag ggaatttcaa taaggatttt tacgacttcg cagtcagtaa
     2701 gggttttttt aaagagggga gctcggttga gttaaaacac ttttttttcg ctcaggatgg
     2761 gaacgctgca atctccgatt acgactacta tcgctacaac ttacctacga tgtgcgatat
     2821 tcgccaatta ctgttcgtag ttgaagtagt agacaagtat ttcgactgtt acgatggagg
     2881 atgtattaat gcgaaccaag taatcgtcaa taatttagat aaatctgcag gttttccctt
     2941 taataagtgg ggaaaggcgc gcctgtacta cgattcaatg tcgtatgagg accaggacgc
     3001 tcttttcgca tataccaaac gcaatgttat cccgacgatt acgcaaatga atctgaaata
     3061 tgccatttcc gctaaaaatc gcgctcgcac ggtcgcggga gtatcgattt gttctactat
     3121 gactaaccgc caattccatc aaaagttgct taaatctatc gcagcaacgc gtggggcaac
     3181 ggtggtcatc gggacctcga agttttatgg aggttggcac aacatgctga aaaccgtgta
     3241 tagtgacgtg gaaaacccgc atctgatggg gtgggattat ccgaaatgcg atcgcgcaat
     3301 gcctaatatg cttcgcatca tggcctcttt agtgctagca cgtaagcata cgacatgttg
     3361 cagcttgtcc catcgctttt accgtttagc taatgagtgc gcacaagtat tatcggagat
     3421 ggtaatgtgc ggcgggagtt tatacgtgaa gccaggtggt actagttctg gggatgcaac
     3481 cacagcgtat gctaattcgg ttttcaatat ctgtcaggcc gttaccgcca acgtaaacgc
     3541 acttctgtca acggatggta ataaaattgc tgataagtac gtacgcaacc tgcaacatcg
     3601 tctgtatgag tgcctttatc gcaaccgcga tgtcgacacc gactttgtaa acgagttcta
     3661 tgcatatttg cgtaagcatt tctctatgat gatcctttcg gccgatgccg ttgtgtgttt
     3721 caacagtacc tatgcctcgc aaggcctagt agcctccatc aaaaacttta agtccgttct
     3781 ttactatcag aacaacgtct ttatgtcaga ggccaagtgc tggaccgaga cggacttaac
     3841 taaaggccca cacgagttct gctcacaaca tactatgctt gtaaagcaag gtgatgacta
     3901 cgtctatctt ccttatcctg acccaagccg catcctggga gctggttgtt tcgttgacga
     3961 tatcgtgaag accgatggca ccctgatgat tgagcgtttc gtctctctgg cgattgacgc
     4021 gtatccgtta actaagcacc ccaatcaaga atacgcggac gtttttcact tatacttaca
     4081 gtatatccgt aagttacacg atgagctgac cggtcacatg cttgatatgt acagcgttat
     4141 gctgacgaat gacaacactt cgcgttattg ggagcctgag ttttatgaag cgatgtatac
     4201 acctcacaca gttctgcagt aagcttgcgg ccgcactcga gtctggtaaa gaaaccgctg
     4261 ctgcgaaatt tgaacgccag cacatggact cgtctactag cgcagcttaa ttaacctagg
     4321 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     4381 ggggtttttt gctgaaacct caggcatttg agaagcacac ggtcacactg cttccggtag
     4441 tcaataaacc ggtaaaccag caatagacat aagcggctat ttaacgaccc tgccctgaac
     4501 cgacgacaag ctgacgaccg ggtctccgca agtggcactt ttcggggaaa tgtgcgcgga
     4561 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     4621 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     4681 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     4741 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     4801 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     4861 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     4921 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     4981 cgcctgagcg agacgaaata cgcggtcgct gttaaaagga caattacaaa caggaatcga
     5041 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     5101 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     5161 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     5221 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     5281 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     5341 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     5401 cctagagcaa gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg
     5461 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     5521 taaacaaata ggcatgcagc gctcttccgc ttcctcgctc actgactcgc tacgctcggt
     5581 cgttcgactg cggcgagcgg tgtcagctca ctcaaaagcg gtaatacggt tatccacaga
     5641 atcaggggat aaagccggaa agaacatgtg agcaaaaagc aaagcaccgg aagaagccaa
     5701 cgccgcaggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     5761 caagccagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     5821 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     5881 tcccttcggg aagcgtggcg ctttctcata gctcacgctg ttggtatctc agttcggtgt
     5941 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     6001 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     6061 cagcagccat tggtaactga tttagaggac tttgtcttga agttatgcac ctgttaaggc
     6121 taaactgaaa gaacagattt tggtgagtgc ggtcctccaa cccacttacc ttggttcaaa
     6181 gagttggtag ctcagcgaac cttgagaaaa ccaccgttgg tagcggtggt ttttctttat
     6241 ttatgagatg atgaatcaat cggtctatca agtcaacgaa cagctattcc gttactctag
     6301 atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag
     6361 ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     6421 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     6481 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6541 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     6601 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     6661 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     6721 g
//
