LOCUS       sequence-473703-        5717 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial protein expression of NSP10.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5717)
  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 5717)
  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-NSP10-SARS-CoV-2
FEATURES             Location/Qualifiers
     source          1..5717
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     544..563
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     628..647
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      758..1346
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1247..1266
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1500..1517
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1532..1671
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1597..1616)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1757..1779
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1773..1964)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2773..3855)
                     /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     3817..3836
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3856..3933)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4139..4158
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     4246..4265
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4246..4264
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4265..4289
                     /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             4304..4326
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             4778..4795
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             4823..4840
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4893..4911)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4907..4954
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4991..5446
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5078..5097)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5288..5309
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5538..5717,1..636))
                     /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(join(5538..5717,1..636))
                     /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"
ORIGIN
        1 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
       61 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
      121 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
      181 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
      241 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
      301 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
      361 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
      421 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
      481 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
      541 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
      601 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
      661 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
      721 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
      781 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
      841 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
      901 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
      961 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     1021 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     1081 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     1141 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     1201 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     1261 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     1321 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     1381 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     1441 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     1501 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     1561 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     1621 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     1681 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     1741 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     1801 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     1861 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     1921 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     1981 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     2041 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     2101 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     2161 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     2221 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     2281 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     2341 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     2401 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     2461 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     2521 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     2581 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     2641 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     2701 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     2761 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     2821 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     2881 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     2941 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     3001 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatgtccgc
     3061 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     3121 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     3181 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     3241 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     3301 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     3361 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     3421 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     3481 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     3541 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     3601 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     3661 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     3721 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     3781 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     3841 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     3901 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     3961 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     4021 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     4081 tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg
     4141 tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga
     4201 tgcgtccggc gtagaggatc gagatcgatc tcgatcccgc gaaattaata cgactcacta
     4261 taggggaatt gtgagcggat aacaattccc ctctagaaat aattttgttt aactttaaga
     4321 aggagatata catatggctg gtaatgctac ggaagtcccg gcaaatagta cggtgctgtc
     4381 gttctgtgcg ttcgctgttg atgcggcaaa agcctacaaa gattatctgg ccagtggcgg
     4441 tcagccgatt acgaactgcg ttaaaatgct gtgtacccat accggtaccg gtcaggccat
     4501 caccgtcacc ccggaagcca atatggacca agaatctttt ggcggtgcat cttgctgtct
     4561 gtattgccgt tgtcatattg atcacccgaa cccgaaaggc ttctgcgacc tgaagggtaa
     4621 atacgtccag atcccgacca cgtgtgctaa cgatccggtg ggctttaccc tgaaaaatac
     4681 ggtgtgcacc gtttgtggca tgtggaaagg ctacggttgc agctgtgacc aactgcgcga
     4741 accgatgctg caaggcggcg gctcaggcgg cggctcacac caccaccacc atcattaatg
     4801 aaagcttgcg gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
     4861 agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
     4921 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggatt
     4981 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     5041 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     5101 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5161 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     5221 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5281 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     5341 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5401 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     5461 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5521 cgctcatgaa ttaattctta gaaaaactca tcgagcatca aatgaaactg caatttattc
     5581 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     5641 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     5701 ccaacatcaa tacaacc
//
