LOCUS       sequence-473702-        6876 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 6876)
  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 6876)
  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-MERS-CoV
FEATURES             Location/Qualifiers
     source          1..6876
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     994..1013
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1033..1110)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1316..1335
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1423..1442
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1423..1441
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1442..1466
                     /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             1481..1503
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3110..3127
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3155..3172
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3225..3243)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3239..3286
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3323..3778
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3410..3429)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3620..3641
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3871..4686)
                     /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(3871..4686)
                     /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     4594..4613
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     4678..4697
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4808..5396
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5297..5316
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5550..5567
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5582..5724
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5650..5669)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5810..5832
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5826..6017)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6826..6876,1..1032))
                     /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 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
       61 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
      121 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
      181 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc
      241 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
      301 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
      361 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
      421 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
      481 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
      541 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
      601 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
      661 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
      721 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
      781 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
      841 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
      901 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
      961 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     1021 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     1081 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     1141 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     1201 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     1261 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     1321 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     1381 gtccggcgta gaggatcgag atcgatctcg atcccgcgaa attaatacga ctcactatag
     1441 gggaattgtg agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg
     1501 agatatacat atgtcacaaa tagtaacagg actatttaaa gattgtagcc gtgagaccag
     1561 cggcttgtcc ccggcgtacg caccgaccta tgtgagcgtt gatgataaat acaagacctc
     1621 tgatgaactt tgcgtgaact tgaatttacc ggctaatgtc ccgtacagtc gtgttattag
     1681 ccgcatgggc ttcaaattag acgctacggt tccgggctac ccgaaactat tcatcacccg
     1741 ggaggaagcg gttcgtcagg ttcgtagctg gatcggtttc gatgtggaag gtgcccacgc
     1801 ctcgcgcaac gcctgcggta caaatgttcc gctgcagctg ggtttcagca ccggtgttaa
     1861 ttttgttgtc caaccagtgg gcgtggtcga caccgagtgg ggtaacatgc tgacgggtat
     1921 cgccgctcgc ccacccccgg gtgaacagtt taagcacctg gttccgctga tgcataaagg
     1981 tgcggcgtgg cctattgtgc gccgtcgtat tgtacaaatg ttgtcggaca cgctggataa
     2041 gttgtccgac tactgtacct tcgtgtgctg ggcacacggc ttcgagctga cctctgcgtc
     2101 ctacttctgc aaaatcggca aagagcagaa atgttgcatg tgtaaccgtc gcgctgctgc
     2161 gtacagcagc ccgctgcaaa gctatgcgtg ctggacccac tcttgcggtt acgattatgt
     2221 ctataacccg ttttttgtcg acgttcaaca gtggggttac gtaggcaacc tggcgactaa
     2281 ccacgatcgt tattgcagcg tgcaccaggg tgcgcacgtg gcctcaaatg atgcgatcat
     2341 gacccgttgc ctggcgattc atagctgctt tatcgagcgc gtggactggg atatcgaata
     2401 cccgtatatc agccatgaaa aaaaactgaa cagctgctgc cgtattgtcg aacgcaatgt
     2461 ggtacgtgca gcgctgctgg cgggtagctt tgacaaggtc tacgatatcg gtaacccgaa
     2521 gggcatcccg attgttgacg atccggttgt ggactggcat tattttgacg cccagccgtt
     2581 gacccgcaaa gtgcaacaac tgttctacac tgaggatatg gcaagccgtt ttgcggatgg
     2641 tctctgcctg ttctggaatt gtaacgttcc gaagtaccca aataacgcaa ttgtttgccg
     2701 ttttgacact cgcgttcaca gcgaattcaa cctgccgggg tgcgacggcg gttcgctgta
     2761 tgtgaataag catgcgtttc ataccccggc gtacgacgtg tctgcgtttc gtgatctgaa
     2821 gccgctcccg tttttctatt actccacgac cccgtgtgaa gttcatggta acggttctat
     2881 gattgaggac atcgactacg tgccgcttaa gagcgcggtt tgcattaccg catgcaactt
     2941 aggaggcgcg gtgtgtcgta agcacgcaac cgagtatcgt gagtatatgg aagcgtacaa
     3001 cttggtttcc gcttccggct tccgcctgtg gtgttacaag acgttcgaca tctataacct
     3061 gtggtccacc ttcaccaaag ttcagggcgg cggctcgggt ggcggcagcc atcaccacca
     3121 tcaccactaa tgaaagcttg cggccgcact cgagcaccac caccaccacc actgagatcc
     3181 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     3241 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     3301 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3361 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3421 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3481 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3541 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3601 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3661 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3721 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttca
     3781 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     3841 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     3901 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     3961 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     4021 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     4081 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     4141 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     4201 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     4261 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     4321 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     4381 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     4441 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     4501 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     4561 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     4621 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     4681 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     4741 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4801 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4861 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4921 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4981 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5041 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5101 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5161 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5221 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5281 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5341 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5401 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5461 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     5521 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     5581 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatatggtg
     5641 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     5701 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     5761 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     5821 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     5881 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     5941 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     6001 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     6061 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     6121 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     6181 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     6241 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     6301 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     6361 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     6421 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     6481 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     6541 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     6601 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     6661 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     6721 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     6781 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     6841 cagtcgggaa acctgtcgtg ccagctgcat taatga
//
