IL263503B2 - DNA polymerase variants from the polX family - Google Patents
DNA polymerase variants from the polX familyInfo
- Publication number
- IL263503B2 IL263503B2 IL263503A IL26350318A IL263503B2 IL 263503 B2 IL263503 B2 IL 263503B2 IL 263503 A IL263503 A IL 263503A IL 26350318 A IL26350318 A IL 26350318A IL 263503 B2 IL263503 B2 IL 263503B2
- Authority
- IL
- Israel
- Prior art keywords
- variant
- nucleic acid
- tdt
- variants
- seq
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1264—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07031—DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR1655475A FR3052462A1 (fr) | 2016-06-14 | 2016-06-14 | Variants d'une adn polymerase de la famille polx |
| PCT/FR2017/051519 WO2017216472A2 (fr) | 2016-06-14 | 2017-06-13 | Variants d'une adn polymérase de la famille polx |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| IL263503A IL263503A (en) | 2019-02-03 |
| IL263503B1 IL263503B1 (en) | 2024-12-01 |
| IL263503B2 true IL263503B2 (en) | 2025-04-01 |
Family
ID=57348782
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| IL263503A IL263503B2 (en) | 2016-06-14 | 2017-06-13 | DNA polymerase variants from the polX family |
Country Status (11)
| Country | Link |
|---|---|
| US (3) | US20200002690A1 (https=) |
| EP (1) | EP3469075A2 (https=) |
| JP (2) | JP7118510B2 (https=) |
| KR (1) | KR102522263B1 (https=) |
| CN (1) | CN109477080A (https=) |
| AU (2) | AU2017286477C1 (https=) |
| CA (1) | CA3024184A1 (https=) |
| FR (2) | FR3052462A1 (https=) |
| IL (1) | IL263503B2 (https=) |
| SG (2) | SG11201809961TA (https=) |
| WO (1) | WO2017216472A2 (https=) |
Families Citing this family (69)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10774316B2 (en) * | 2014-10-20 | 2020-09-15 | Molecular Assemblies, Inc. | Modified template-independent enzymes for polydeoxynucleotide synthesis |
| AU2018272302A1 (en) * | 2017-05-26 | 2019-12-19 | Nuclera Nucleics Ltd | Use of terminal transferase enzyme in nucleic acid synthesis |
| US11390856B2 (en) | 2017-08-07 | 2022-07-19 | Dna Script | Variants of family a DNA polymerase and uses thereof |
| US10752887B2 (en) | 2018-01-08 | 2020-08-25 | Dna Script | Variants of terminal deoxynucleotidyl transferase and uses thereof |
| US10435676B2 (en) | 2018-01-08 | 2019-10-08 | Dna Script | Variants of terminal deoxynucleotidyl transferase and uses thereof |
| EP3826762A1 (en) | 2018-07-23 | 2021-06-02 | DNA Script | Massively parallel enzymatic synthesis of nucleic acid strands |
| CA3119845A1 (en) | 2018-11-14 | 2020-05-22 | Dna Script | Terminal deoxynucleotidyl transferase variants and uses thereof |
| SG11202105441WA (en) | 2018-12-13 | 2021-06-29 | Dna Script | Direct oligonucleotide synthesis on cells and biomolecules |
| WO2020141143A1 (en) | 2019-01-03 | 2020-07-09 | Dna Script | One pot synthesis of sets of oligonucleotides |
| GB201901501D0 (en) * | 2019-02-04 | 2019-03-27 | Nuclera Nucleics Ltd | Modified terminal deoxynucleotidyl transferase (TdT) enzymes |
| WO2020165137A1 (en) | 2019-02-12 | 2020-08-20 | Dna Script | Efficient product cleavage in template-free enzymatic synthesis of polynucleotides. |
| EP4407026A3 (en) * | 2019-05-28 | 2025-01-15 | DNA Script | Variants of terminal deoxynucleotidyl transferase and uses thereof |
| EP3744854A1 (en) * | 2019-05-28 | 2020-12-02 | DNA Script | Variants of terminal deoxynucleotidyl transferase and uses thereof |
| WO2021018919A1 (en) | 2019-07-30 | 2021-02-04 | Dna Script | Template-free enzymatic synthesis of polynucleotides using poly(a) and poly(u) polymerases |
| JP2022543568A (ja) | 2019-08-01 | 2022-10-13 | ディーエヌエー スクリプト | ポリヌクレオチドの鋳型なしの酵素による合成における長い配列の収量の増加 |
| CN110331136B (zh) * | 2019-09-05 | 2019-12-24 | 中国科学院天津工业生物技术研究所 | 一种末端脱氧核糖核苷转移酶变体及其应用 |
| CN114555818A (zh) | 2019-09-09 | 2022-05-27 | Dna斯克瑞普特公司 | 使用光可切割连接的无模板酶促多核苷酸合成 |
| WO2021058438A1 (en) | 2019-09-23 | 2021-04-01 | Dna Script | Increasing long-sequence yields in template-free enzymatic synthesis of polynucleotides |
| AU2020384271A1 (en) | 2019-11-13 | 2022-06-23 | Dna Script | High efficiency template-free enzymatic synthesis of polynucleotides |
| CN114787346A (zh) * | 2019-12-12 | 2022-07-22 | Dna斯克瑞普特公司 | 用于多核苷酸无模板酶学合成的嵌合末端脱氧核苷酸转移酶 |
| WO2021122539A1 (en) | 2019-12-16 | 2021-06-24 | Dna Script | Template-free enzymatic polynucleotide synthesis using dismutationless terminal deoxynucleotidyl transferase variants |
| EP4110940B1 (en) | 2020-02-25 | 2024-03-27 | DNA Script | Method and apparatus for enzymatic synthesis of polynucleotides |
| US20230159903A1 (en) | 2020-04-20 | 2023-05-25 | Dna Script | Terminal Deoxynucleotidyl Transferase Variants and Uses Thereof |
| JP7828081B2 (ja) * | 2020-05-12 | 2026-03-11 | ナショナル ユニヴァーシティー オブ シンガポール | 熱安定性末端デオキシヌクレオチジルトランスフェラーゼ |
| EP4150065A2 (en) | 2020-05-12 | 2023-03-22 | Illumina Inc | Generating nucleic acids with modified bases using recombinant terminal deoxynucleotidyl transferase |
| CN116075593A (zh) * | 2020-06-03 | 2023-05-05 | 分子组装公司 | 用于聚脱氧核苷酸合成的经修饰的非模板依赖性酶 |
| CN116249782A (zh) | 2020-06-12 | 2023-06-09 | 辛希克斯 | 使用热稳定性酶从头开始的不依赖于模板的核酸合成 |
| WO2021254934A1 (en) | 2020-06-16 | 2021-12-23 | Dna Script | Systems, apparatus and kits for enzymatic polynucleotide synthesis |
| WO2022013094A1 (en) | 2020-07-15 | 2022-01-20 | Dna Script | Massively parallel enzymatic synthesis of polynucleotides |
| US11993801B2 (en) | 2020-07-21 | 2024-05-28 | Illumina Singapore Pte. Ltd. | Base-modified nucleotides as substrates for TdT-based enzymatic nucleic acid synthesis |
| WO2022029427A1 (en) * | 2020-08-04 | 2022-02-10 | Nuclera Nucleics Ltd | MODIFIED TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE (TdT) ENZYMES |
| GB2598152B (en) * | 2020-08-21 | 2025-04-16 | Nuclera Ltd | Modified terminal deoxynucleotidyl transferase (TdT) enzymes |
| AU2021347452A1 (en) | 2020-09-22 | 2023-04-27 | Dna Script | Stabilized n-terminally truncated terminal deoxynucleotidyl transferase variants and uses thereof |
| EP4237573A1 (en) | 2020-10-29 | 2023-09-06 | DNA Script | Enzymatic synthesis of polynucleotide probes |
| US12391970B2 (en) | 2020-11-11 | 2025-08-19 | Microsoft Technology Licensing, Llc | Spatial control of polynucleotide synthesis by strand capping |
| CN112322715B (zh) * | 2020-11-17 | 2022-11-25 | 清华大学 | 一种核酸测序方法 |
| FR3121442A1 (fr) | 2021-04-02 | 2022-10-07 | Dna Script | Procédés et trousses pour la synthèse enzymatique de polynucléotides pouvant former des g4 |
| NL2032097B1 (en) | 2021-06-10 | 2024-03-29 | Dna Script | Enzymatic synthesis of polynucleotides using 3'-o-amino-2'-deoxyribonucleoside triphosphate monomers |
| EP4392554A1 (en) | 2021-08-23 | 2024-07-03 | DNA Script | Method for generating double-stranded nucleic acid |
| US20240299945A1 (en) | 2021-09-03 | 2024-09-12 | Elegen Corporation | Multi-way bead-sorting devices, systems, and methods of use thereof using pressure sources |
| CA3234383A1 (en) | 2021-10-15 | 2023-04-27 | Codexis, Inc. | Engineered terminal deoxynucleotidyl transferase variants |
| WO2023083997A2 (en) | 2021-11-10 | 2023-05-19 | Dna Script | Novel terminal deoxynucleotidyl |
| EP4430176A2 (en) | 2021-11-10 | 2024-09-18 | DNA Script | Novel terminal deoxynucleotidyl transferase (tdt) variants |
| CN116555216A (zh) * | 2022-01-28 | 2023-08-08 | 中国科学院天津工业生物技术研究所 | 可控合成单链dna的末端转移酶变体及应用 |
| WO2023170286A2 (en) | 2022-03-11 | 2023-09-14 | Dna Script | Alignment post and secure mechanism for enzymatic polynucleotide synthesis |
| US20250205700A1 (en) | 2022-03-11 | 2025-06-26 | Dna Script | Automation station for enzymatic polynucleotide synthesis |
| WO2023170258A1 (en) | 2022-03-11 | 2023-09-14 | Dna Script | Apparatus for enzymatic synthesis of a plurality of polynucleotides comprising a condensation trap |
| WO2023170259A1 (en) | 2022-03-11 | 2023-09-14 | Dna Script | Modular accessory rack |
| WO2023240202A2 (en) * | 2022-06-08 | 2023-12-14 | Ansa Biotechnologies, Inc. | Modified terminal deoxynucleotidyl transferase polymerases |
| EP4555083A1 (en) | 2022-07-13 | 2025-05-21 | DNA Script | Method of purifying solution comprising polynucleotide |
| EP4630540A1 (en) | 2022-12-05 | 2025-10-15 | DNA Script | Variants of poly(a) polymerase and uses thereof |
| CN120359296A (zh) | 2022-12-09 | 2025-07-22 | Dna斯克瑞普特公司 | 体外酶促rna合成 |
| WO2024141628A1 (en) | 2022-12-31 | 2024-07-04 | Dna Script | Variable viscosity inks for inkjet delivery of enzyme reagents |
| WO2024153642A1 (en) | 2023-01-16 | 2024-07-25 | Dna Script | Scarless template-free enzymatic synthesis of polynucleotides |
| WO2024153643A1 (en) | 2023-01-16 | 2024-07-25 | Dna Script | Inkjet-assisted enzymatic nucleic acid synthesis |
| CN120530202A (zh) | 2023-01-26 | 2025-08-22 | Dna斯克瑞普特公司 | 多核苷酸探针的酶促合成 |
| WO2024227846A2 (en) | 2023-05-03 | 2024-11-07 | Dna Script | Reagent used in deprotection of 3'-o-amino polynucleotides |
| WO2024241324A1 (en) * | 2023-05-24 | 2024-11-28 | Scintium Ltd | Method for training an artificial intelligence (ai) model and generating a 3d computer-aided design (cad) and system therefor |
| WO2024256604A1 (en) | 2023-06-15 | 2024-12-19 | Dna Script | Salt addition during enzymatic polynucleotide synthesis |
| EP4735621A1 (en) | 2023-06-30 | 2026-05-06 | DNA Script | Nucleic acid synthesis on reusable support |
| CN121532522A (zh) | 2023-07-27 | 2026-02-13 | Dna斯克瑞普特公司 | 用于产生多个多核苷酸的方法 |
| WO2025088024A1 (en) | 2023-10-24 | 2025-05-01 | Dna Script | Novel terminal deoxynucleotidyl transferase (tdt) variants and uses thereof |
| WO2025093659A1 (en) | 2023-11-04 | 2025-05-08 | Dna Script | 3'-o-aminooxymethyl blocked nucleotides and use thereof in enzymatic synthesis of polynucleotides |
| WO2025137872A1 (zh) * | 2023-12-26 | 2025-07-03 | 华大工程生物学长荡湖研究所 | 一种末端脱氧核糖核苷转移酶突变体及其制备方法和应用 |
| WO2025155239A1 (en) * | 2024-01-17 | 2025-07-24 | Agency For Science, Technology And Research | X family dna polymerases and uses thereof |
| EP4653525A1 (en) * | 2024-05-21 | 2025-11-26 | Uab "Biomatter Designs" | Engineered terminal deoxynucleotidyl transferase polymerases |
| WO2026008717A2 (en) | 2024-07-02 | 2026-01-08 | Dna Script | Method for enzymatic synthesis of polynucleotides |
| WO2026008711A2 (en) | 2024-07-02 | 2026-01-08 | Dna Script | Filtering device and method for polynucleotide enzymatic synthesis |
| WO2026017834A1 (en) | 2024-07-19 | 2026-01-22 | Dna Script | Inkjet-assisted enzymatic nucleic acid synthesis and cleavage |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016064880A1 (en) * | 2014-10-20 | 2016-04-28 | Molecular Assemblies, Inc. | Modified template-independent enzymes for polydeoxynucleotide systhesis |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5004689A (en) | 1982-02-22 | 1991-04-02 | Biogen, Massachusetts | DNA sequences, recombinant DNA molecules and processes for producing human gamma interferon-like polypeptides in high yields |
| IL71691A (en) | 1984-04-27 | 1991-04-15 | Yeda Res & Dev | Production of interferon-ypsilon |
| US5047335A (en) | 1988-12-21 | 1991-09-10 | The Regents Of The University Of Calif. | Process for controlling intracellular glycosylation of proteins |
| BG52073B2 (en) | 1990-01-24 | 1996-04-30 | Inst Molekuljarna Biolog | Method for the preparation of recombinant human noncystein -interferon, free of n-end methionine |
| US6812339B1 (en) * | 2000-09-08 | 2004-11-02 | Applera Corporation | Polymorphisms in known genes associated with human disease, methods of detection and uses thereof |
| CA2742593C (en) * | 2008-11-03 | 2020-05-05 | Kapa Biosystems, Inc. | Chimeric dna polymerases |
| CA2864394C (en) * | 2011-03-02 | 2021-10-19 | Jack Roth | A method of predicting a response to a tusc2 therapy |
| EP3080158A4 (en) * | 2013-12-09 | 2018-03-21 | Adimab, LLC | Polyclonal mixtures of antibodies, and methods of making and using them |
| FR3020071B1 (fr) | 2014-04-17 | 2017-12-22 | Dna Script | Procede de synthese d'acides nucleiques, notamment d'acides nucleiques de grande longueur, utilisation du procede et kit pour la mise en œuvre du procede |
| FR3025201B1 (fr) | 2014-09-02 | 2018-10-12 | Dna Script | Nucleotides modifies pour la synthese d'acides nucleiques, un kit renfermant de tels nucleotides et leur utilisation pour la production de genes ou sequences d'acides nucleiques synthetiques |
-
2016
- 2016-06-14 FR FR1655475A patent/FR3052462A1/fr active Pending
-
2017
- 2017-06-13 IL IL263503A patent/IL263503B2/en unknown
- 2017-06-13 SG SG11201809961TA patent/SG11201809961TA/en unknown
- 2017-06-13 JP JP2018564828A patent/JP7118510B2/ja active Active
- 2017-06-13 WO PCT/FR2017/051519 patent/WO2017216472A2/fr not_active Ceased
- 2017-06-13 CA CA3024184A patent/CA3024184A1/en active Pending
- 2017-06-13 CN CN201780037427.0A patent/CN109477080A/zh active Pending
- 2017-06-13 KR KR1020187034921A patent/KR102522263B1/ko active Active
- 2017-06-13 US US16/309,414 patent/US20200002690A1/en not_active Abandoned
- 2017-06-13 EP EP17746156.3A patent/EP3469075A2/fr active Pending
- 2017-06-13 AU AU2017286477A patent/AU2017286477C1/en active Active
- 2017-06-13 SG SG10202009688QA patent/SG10202009688QA/en unknown
- 2017-07-07 FR FR1756408A patent/FR3053699B1/fr active Active
-
2022
- 2022-06-30 JP JP2022105854A patent/JP2022137127A/ja active Pending
- 2022-07-27 US US17/815,490 patent/US20230167421A1/en active Pending
-
2023
- 2023-11-21 US US18/516,616 patent/US20240124855A1/en active Pending
- 2023-12-13 AU AU2023282219A patent/AU2023282219A1/en not_active Abandoned
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016064880A1 (en) * | 2014-10-20 | 2016-04-28 | Molecular Assemblies, Inc. | Modified template-independent enzymes for polydeoxynucleotide systhesis |
Non-Patent Citations (1)
| Title |
|---|
| YANG, BAOLI, KAREN N. GATHY, AND MARY SUE COLEMAN., T-CELL SPECIFIC AVIAN TDT: CHARACTERIZATION OF THE CDNA AND RECOMBINANT ENZYME., 11 June 1995 (1995-06-11) * |
Also Published As
| Publication number | Publication date |
|---|---|
| IL263503A (en) | 2019-02-03 |
| IL263503B1 (en) | 2024-12-01 |
| AU2023282219A1 (en) | 2024-01-18 |
| US20230167421A1 (en) | 2023-06-01 |
| SG10202009688QA (en) | 2020-11-27 |
| JP7118510B2 (ja) | 2022-08-16 |
| CA3024184A1 (en) | 2017-12-21 |
| WO2017216472A3 (fr) | 2018-03-15 |
| FR3053699A1 (fr) | 2018-01-12 |
| FR3052462A1 (fr) | 2017-12-15 |
| US20200002690A1 (en) | 2020-01-02 |
| AU2017286477B2 (en) | 2023-09-14 |
| CN109477080A (zh) | 2019-03-15 |
| SG11201809961TA (en) | 2018-12-28 |
| JP2022137127A (ja) | 2022-09-21 |
| AU2017286477C1 (en) | 2023-12-21 |
| KR20190024883A (ko) | 2019-03-08 |
| EP3469075A2 (fr) | 2019-04-17 |
| JP2019517799A (ja) | 2019-06-27 |
| AU2017286477A1 (en) | 2018-12-06 |
| FR3053699B1 (fr) | 2021-04-02 |
| KR102522263B1 (ko) | 2023-04-18 |
| WO2017216472A2 (fr) | 2017-12-21 |
| US20240124855A1 (en) | 2024-04-18 |
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