LOCUS       sequence-328208-        6113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of  codon optimized SARS-COV-2 Nsp8 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6113)
  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 6113)
  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: pIA1363
FEATURES             Location/Qualifiers
     source          1..6113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      30..485
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(117..136)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     327..348
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(577..1392)
                     /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(577..1392)
                     /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     1300..1319
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1384..1403
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1514..2102
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2003..2022
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2256..2273
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2288..2427
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2353..2372)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2513..2535
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2529..2720)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3529..4611)
                     /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     4573..4592
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4612..4689)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4895..4914
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4998..5017
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4998..5016
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5017..5041
                     /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             5056..5078
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5098..5121
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             5290..5310
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5975..5992
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6045..6063)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6059..6106
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
       61 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
      121 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
      181 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
      241 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
      301 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
      361 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
      421 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
      481 aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
      541 atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat cgagcatcaa
      601 atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa aaagccgttt
      661 ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat cctggtatcg
      721 gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct cgtcaaaaat
      781 aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga atggcaaaag
      841 tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt catcaaaatc
      901 actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac gaaatacgcg
      961 atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca ggaacactgc
     1021 cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct ggaatgctgt
     1081 tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga taaaatgctt
     1141 gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct catctgtaac
     1201 atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat cgggcttccc
     1261 atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc atttataccc
     1321 atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg tttcccgttg
     1381 aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca
     1441 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1501 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     1561 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     1621 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     1681 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     1741 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     1801 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     1861 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     1921 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     1981 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2041 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     2101 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     2161 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     2221 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     2281 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     2341 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     2401 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     2461 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     2521 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     2581 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     2641 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     2701 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     2761 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     2821 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     2881 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     2941 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     3001 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     3061 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     3121 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     3181 acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     3241 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     3301 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     3361 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     3421 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     3481 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     3541 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3601 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     3661 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     3721 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     3781 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     3841 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     3901 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     3961 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     4021 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     4081 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     4141 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     4201 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     4261 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     4321 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     4381 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     4441 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     4501 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     4561 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     4621 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     4681 gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca
     4741 gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg
     4801 cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca
     4861 tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag
     4921 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga
     4981 tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg ataacaattc
     5041 ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatggg cagcagccat
     5101 caccatcacc atcatcacca tggagagtat aaactggcac tgaatggtaa aaccctgaaa
     5161 ggtgaaacca ccaccgaagc agttgatgca gcaaccgcag aaaaagtctt taaacagtat
     5221 gccaatgata atggcgttga tggtgaatgg acctatgatg atgcaaccaa aacctttacc
     5281 gtgaccgaag aaaatctgta tttccaggga tccgcaatcg cctctgaatt ttcctccctt
     5341 ccatcttacg cagcctttgc gaccgcacaa gaggcatacg agcaggccgt agcaaacgga
     5401 gacagcgagg tcgttttaaa gaagttgaaa aagtctttaa atgtggctaa gtctgaattt
     5461 gaccgcgatg ctgccatgca acgcaaattg gagaaaatgg cggatcaagc tatgacccag
     5521 atgtataagc aagcacgttc agaagacaaa cgtgcgaaag tcacctcggc gatgcagact
     5581 atgttattca ccatgttgcg caaattagat aacgatgcct taaacaacat cattaacaac
     5641 gcccgtgatg gatgtgttcc gctgaacatc atccccctga cgactgcggc gaaacttatg
     5701 gtagttatcc ctgattacaa tacatataag aacacttgtg acggtactac cttcacgtat
     5761 gccagcgcgt tatgggaaat ccaacaagtg gttgatgcag attcaaagat tgtgcagctc
     5821 tctgaaatct cgatggacaa ttcgccgaat ctggcatggc cccttatcgt tacggccctg
     5881 cgtgcaaatt cagccgtgaa acttcaataa gcttatcgga tcccgggccc gtcgactgca
     5941 gaggcctgca tgcaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc
     6001 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     6061 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aag
//
