LOCUS       sequence-473706-        6885 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial protein expression of NSP14.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6885)
  AUTHORS   Rona G, Zeke A, Miwatani-Minter B, de Vries M, Kaur R, Schinlever A,
            Garcia SF, Goldberg HV, Wang H, Hinds TR, Bailly F, Zheng N, Cotelle
            P, Desmaele D, Landau NR, Dittmann M, Pagano M
  TITLE     The NSP14/NSP10 RNA repair complex as a Pan-coronavirus therapeutic 
            target.
  JOURNAL   Cell Death Differ. 2022 Feb;29(2):285-292. doi: 
            10.1038/s41418-021-00900-1. Epub 2021 Dec 3.
  PUBMED    34862481
REFERENCE   2  (bases 1 to 6885)
  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"; doi: 
            "10.1038/s41418-021-00900-1"; journalName: "Cell Death Differ"; 
            date: "2022-02"; volume: "29"; issue: "2"; pages: "285-292"
COMMENT     Sequence Label: pET-30a-NSP14-SARS-CoV
FEATURES             Location/Qualifiers
     source          1..6885
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        189..266
                     /gene="lacI"
                     /label=lacI promoter
     CDS             267..1349
                     /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     complement(286..305)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2158..2349
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2343..2365)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2451..2593
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2506..2525
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2608..2625)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2779..3367)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2859..2878)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3478..3497)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3489..4304
                     /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             3489..4304
                     /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     complement(3562..3581)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4397..4852)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4534..4555)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4746..4765
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4889..4936)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4932..4950
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5003..5020)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5048..5065)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6681..6703
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6718..6742
                     /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)."
     primer_bind     complement(6742..6761)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6743..6761)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6849..6868)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 tcgccacttc gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc
       61 cgggggactg ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa
      121 cggcctcaac ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg
      181 agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg
      241 aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt
      301 atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg
      361 cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg
      421 tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg
      481 ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg
      541 ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc
      601 acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg
      661 atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg
      721 accagacacc catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg
      781 agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg
      841 tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc
      901 cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa
      961 tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg
     1021 gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggacatc tcggtagtgg
     1081 gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg
     1141 attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg
     1201 cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc
     1261 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     1321 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact
     1381 cattaggcac cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc
     1441 cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa
     1501 ctcgtaggac aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg
     1561 agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa
     1621 gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc
     1681 atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt tgaggacccg gctaggctgg
     1741 cggggttgcc ttactggtta gcagaatgaa tcaccgatac gcgagcgaac gtgaagcgac
     1801 tgctgctgca aaacgtctgc gacctgagca acaacatgaa tggtcttcgg tttccgtgtt
     1861 tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca ttatgttccg gatctgcatc
     1921 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat
     1981 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca
     2041 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct
     2101 cgtttcatcg gtatcattac ccccatgaac agaaatcccc cttacacgga ggcatcagtg
     2161 accaaacagg aaaaaaccgc ccttaacatg gcccgcttta tcagaagcca gacattaacg
     2221 cttctggaga aactcaacga gctggacgcg gatgaacagg cagacatctg tgaatcgctt
     2281 cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc gtttcggtga tgacggtgaa
     2341 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     2401 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
     2461 acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
     2521 ttgtactgag agtgcaccat atatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2581 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     2641 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     2701 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2761 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2821 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2881 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2941 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3001 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3061 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3121 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3181 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     3241 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3301 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3361 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3421 acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa
     3481 ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca
     3541 acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg
     3601 cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca
     3661 aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat
     3721 ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca
     3781 ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg
     3841 aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta
     3901 attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata
     3961 acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag
     4021 tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg
     4081 atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg
     4141 gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg
     4201 agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata
     4261 tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc
     4321 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4381 cgaaaagtgc cacctgaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt
     4441 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     4501 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     4561 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     4621 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     4681 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     4741 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     4801 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     4861 ccaatccgga tatagttcct cctttcagca aaaaacccct caagacccgt ttagaggccc
     4921 caaggggtta tgctagttat tgctcagcgg tggcagcagc caactcagct tcctttcggg
     4981 ctttgttagc agccggatct cagtggtggt ggtggtggtg ctcgagtgcg gccgcaagct
     5041 ttcattagtg gtggtggtgg tggtgggagc cgccaccaga gccgccacct tgcaggcggg
     5101 taaaggtgtt ccataagttg tacgtgtcga attgcttata aatccacaga gagaaacccg
     5161 cgctaatcat catgttatac gcatccagat actggcggta ttcattcgcg tgatggcggc
     5221 acacggcacc gcccaagtta caacgggtga tgcatgtcgc gctcttcaac ggcacgtagt
     5281 cgatgtcgct aacgacttgt ttaccatggc tttcgcacgg gctgtcgcta taataaaaaa
     5341 acggcagttg cttgagattg gtgaaggctg acttatcgaa tgccggggta tggaacgcat
     5401 gtttgttcac atacagcgaa ccgccgtcgc acccgggtaa attcaaatta gacaacacgc
     5461 gggtatcaaa gcgacaaaca atggcattcg ccggataacg gtcgacgttg cagttccaaa
     5521 acaagcacac accatcggta aacttatcgt gatgcgtagc ataactgtag aacaactctt
     5581 caatcttgta cgccttgtcg ctgcacggct gcgcatcgta aaatttccac tcaacttccg
     5641 cctgcggtac gcacttgatt gctttcgggt tgccgatgtc gtgcagaacc gggaacttat
     5701 cggccaggag cgcggacttc acaaccatgt gctggacttt acggcacgcg ctgttcacac
     5761 gcagttcgtc gccgataatc gggtactcga cggaccagtc aacacgttta acaaagcact
     5821 cgtggaccgc caggcaacga gtcatgatcg catcacagct ggcaacatgc gcgttgccgt
     5881 gaacctgaca atgctgatcg tggttgcttt gcaggttacc ggtgaaaccc cactgctgca
     5941 catcgatcat aaatgggtta tacacgtagt cgaagcctac gctgtggttc caacaggcgt
     6001 aagtgtcgct agaggtgcta aagcacgtcg cgcgcttatc acacagacag caggtacgct
     6061 ccggaccgat tttcacgaag tatttcatgc tggtcaattc gaagccatgt gcccacagca
     6121 caaagacaac gcggtcgcta agacccttca gggtgtcgga cagcatttgt acgatcttaa
     6181 tacgcacaac attccacggc agacctttat acatcaacgg gatcaggtgt ttaaactggt
     6241 cgcccggagg cggtttcgca ttaactcggg tgaactcggt gttgttctca gtgtccacgt
     6301 agcccgttgg gacagcaacc aagttcaccc cggtcgagaa acccagttgc agtggaaggt
     6361 ttgtacccac agcatctctg gtagcatggc agccttcaac gtcgaatcca atccaagcac
     6421 gaacatgacg aattgcctct tcacgcgtga tgaacatatt cgggtaacca ttaacctgat
     6481 aattcatctt gaaacccatc atagaaatca gacgacggta ggtcatatct ttcggaatac
     6541 ccggaatatc cacgcacaga ccctcggttt taaatttgat atccacggac aggtgagtcg
     6601 gtgcctgggt cggatgcagg ccggtgatga ttttggaaca gtctttaaat agtcctgtta
     6661 cattttcagc catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga
     6721 attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc
     6781 gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
     6841 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggc
//
