LOCUS       sequence-338007-        5811 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of SARS-COV-2 Nsp9 (extra GS at the N-terminal) in E. 
            coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5811)
  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 5811)
  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: pIA1364
FEATURES             Location/Qualifiers
     source          1..5811
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      10..465
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(97..116)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     307..328
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(557..1372)
                     /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(557..1372)
                     /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     1280..1299
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1364..1383
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1494..2082
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1983..2002
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2236..2253
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2268..2407
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2333..2352)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2493..2515
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2509..2700)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3509..4591)
                     /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"
     primer_bind     4553..4572
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4592..4669)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4875..4894
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4978..4997
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4978..4996
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4997..5021
                     /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             5036..5058
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5078..5101
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             5270..5290
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5655..5672
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5725..5743)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5739..5786
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
       61 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
      121 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
      181 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
      241 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
      301 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
      361 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
      421 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
      481 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
      541 gctcatgaat taattcttag aaaaactcat cgagcatcaa atgaaactgc aatttattca
      601 tatcaggatt atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact
      661 caccgaggca gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc
      721 caacatcaat acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat
      781 caccatgagt gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga
      841 cttgttcaac aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt
      901 tattcattcg tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat
      961 tacaaacagg aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt
     1021 cacctgaatc aggatattct tctaatacct ggaatgctgt tttcccgggg atcgcagtgg
     1081 tgagtaacca tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa
     1141 attccgtcag ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt
     1201 tgccatgttt cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg
     1261 cacctgattg cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt
     1321 tggaatttaa tcgcggccta gagcaagacg tttcccgttg aatatggctc ataacacccc
     1381 ttgtattact gtttatgtaa gcagacagtt ttattgttca tgaccaaaat cccttaacgt
     1441 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     1501 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     1561 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     1621 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     1681 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     1741 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     1801 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     1861 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     1921 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     1981 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2041 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2101 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     2161 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     2221 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     2281 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     2341 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     2401 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     2461 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     2521 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     2581 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     2641 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     2701 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     2761 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     2821 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     2881 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     2941 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac
     3001 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     3061 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     3121 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc
     3181 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     3241 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     3301 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     3361 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     3421 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     3481 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3541 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3601 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     3661 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     3721 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     3781 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     3841 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     3901 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     3961 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     4021 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     4081 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     4141 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     4201 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     4261 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     4321 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     4381 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     4441 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     4501 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     4561 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     4621 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     4681 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     4741 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     4801 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     4861 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     4921 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
     4981 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
     5041 ttaactttaa gaaggagata tacatatggg cagcagccat caccatcacc atcatcacca
     5101 tggagagtat aaactggcac tgaatggtaa aaccctgaaa ggtgaaacca ccaccgaagc
     5161 agttgatgca gcaaccgcag aaaaagtctt taaacagtat gccaatgata atggcgttga
     5221 tggtgaatgg acctatgatg atgcaaccaa aacctttacc gtgaccgaag aaaatctgta
     5281 tttccaggga tccaacaacg aactttctcc ggtcgcgttg cgtcaaatgt cttgcgctgc
     5341 tggcaccacc caaacggcgt gtacggatga caacgcctta gcatactata atactacaaa
     5401 aggtggccgt ttcgtgcttg ctttgttatc tgatttacag gatttgaaat gggcacgttt
     5461 tcctaagagt gacggaacgg gaaccattta tacggaatta gagcctccgt gccgtttcgt
     5521 caccgataca ccaaaaggcc cgaaagtgaa gtacctgtac ttcatcaagg gtctgaataa
     5581 tctgaatcgc ggcatggttc tgggtagttt ggctgcgacc gtgcgtctgc aataagcttg
     5641 cggccgcact cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa
     5701 aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct
     5761 ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga t
//
