LOCUS       sequence-473701-        6881 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 6881)
  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 6881)
  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-2
FEATURES             Location/Qualifiers
     source          1..6881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        190..267
                     /gene="lacI"
                     /label=lacI promoter
     CDS             268..1350
                     /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(287..306)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2159..2350
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(2344..2366)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2452..2591
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2507..2526
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2606..2623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2777..3365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2857..2876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(3476..3495)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3487..4302
                     /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             3487..4302
                     /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(3560..3579)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(4394..4849)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4531..4552)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4743..4762
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4886..4933)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4929..4947
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(5000..5017)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(5045..5062)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6678..6700
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6715..6739
                     /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(6739..6758)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6740..6758)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6846..6865)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
       61 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
      121 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
      181 gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
      241 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
      301 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
      361 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
      421 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
      481 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
      541 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
      601 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
      661 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
      721 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
      781 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
      841 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
      901 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
      961 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
     1021 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggacat ctcggtagtg
     1081 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     1141 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     1201 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     1261 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     1321 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     1381 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
     1441 ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca
     1501 actcgtagga caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg
     1561 gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca
     1621 agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg
     1681 catggcggcc ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     1741 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     1801 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     1861 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     1921 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     1981 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     2041 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     2101 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt
     2161 gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac
     2221 gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
     2281 tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga
     2341 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     2401 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     2461 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     2521 attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat
     2581 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     2641 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     2701 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     2761 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     2821 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2881 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     2941 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     3001 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     3061 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     3121 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     3181 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     3241 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     3301 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     3361 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     3421 gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg
     3481 ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac
     3541 atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg
     3601 acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa
     3661 ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt
     3721 atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc
     3781 actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa
     3841 aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat
     3901 tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac
     3961 ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc
     4021 tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat
     4081 ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga
     4141 cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag
     4201 ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg
     4261 aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg
     4321 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4381 aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     4441 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     4501 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     4561 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     4621 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     4681 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     4741 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     4801 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca
     4861 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
     4921 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
     4981 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagctttc
     5041 attaatggtg atggtggtgg tgggagccgc cgccagaacc gccgccttgc agacgggtaa
     5101 acgtattcca caggttgtag gtatcaaact gtttatacac ccacagagag aaacccgcgc
     5161 taatcatcat gttgtaagca tccaggtaca ggcgatattc attcgcgtga tgacgacaaa
     5221 cagcaccgcc caggttacaa cgcgtgatgc aggtcgcact tttcagcggc acatagtcaa
     5281 tatcagaaac cacttgtttg ccatggcttt cgcacggaga atcgctgtaa tagaaaaacg
     5341 gcagctgttt caggttaaca aacgcggatt tgtcgaaagc cggggtgtga aatgcatgtt
     5401 tattgacgta cagtgaaccg ccatcacaac ccggcagatt caggttggac aggacgcggg
     5461 tgtcaaaacg gcaaacgata ctattggccg gatagcgatc gacattacag ttccaaaaca
     5521 ggcaaacgcc gtcggtgaat ttatcggaat gcgtagcgta actataaaac agttcttcga
     5581 ttttgtatgc tttgtcagaa cacggttggg catcatagaa tttccattca acatccgcct
     5641 gcggcacgca tttgatagct ttcgggttac caatgtcgtg cagaaccgga aatttatcgg
     5701 ccagcagagc tgctttgaca accatgtgct gcactttacg acaggccgca ttgattttca
     5761 gttcgtcgcc gataatcgga tattcaatgg tccaatcgac gcgtttaaca aaacattcgt
     5821 gcactgccag gcaacgcgtc ataatggcgt cacagctggc cacgtgcgcg ttaccatgga
     5881 cttggcaata cagatcatgg ttcgactgca gattaccggt gaagccccat tgctgcacgt
     5941 cgatcataaa cggattgtag acataatcga agccaatact gtgatgccag caggcgtagg
     6001 tgtccgacgc cgttgaaaaa caggtcgcgc gacgatcgca cagacagcag gtgcgttccg
     6061 gaccgatttt gacgaaatat ttcatagacg tcagttcaaa gccgtgtgcc cacagaacga
     6121 acacgacgcg gtccgacagg tttttcaggg tatctgacag catttgaacg attttaatac
     6181 gaaccacatt ccacggcaga cctttgtaca tcagcggaat cagatgttta aactgatcac
     6241 ccggcggcgg ttttgcggac acgcgactga agtccgtatt gttcggggta tcaacataac
     6301 cggtcggcac cgcaaccagg ttcacacccg tgctaaagcc cagctgcagc ggcagattgg
     6361 tgccaacagc ttcgcgcgtt gcgtgacaac cttcaacatc gaagccgatc catgcgcgca
     6421 catgacggat ggcttcttca cgggtaataa acatgttcgg gtaaccattc acttgatagt
     6481 tcattttgaa gcccatcatg ctaatcaggc gacgatacgt catgtctttc gggatacccg
     6541 gaatatccac gcacaggcct tccgttttaa atttggtatc gacagacagg tgcgtcggtg
     6601 cttgcgtcgg atgcagaccc gtgataactt tggaacaatc tttgaacaga cccgtgacat
     6661 tttcggccat atgtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt
     6721 gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatcgat
     6781 ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc
     6841 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg g
//
