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application/pdf PDFMetadata: Objects: 598 FreeObjects: 4 IncrementalUpdates: 2 DocumentCatalog: PageLayout: SinglePage PageMode: UseOutlines Outlines: Item: Title: A novel MoClo-mediated intron insertion system facilitates enhanced transgene expression in Chlamydomonas reinhardtii Destination: bmark_-1739839278 Children: Item: Title: 1 Introduction Destination: s1 Item: Title: 2 Materials and methods Destination: s2 Children: Item: Title: 2.1 Chlamydomonas reinhardtii cultivation and transformation Destination: s2_1 Item: Title: 2.2 Transgene design and cloning Destination: s2_2 Item: Title: 2.3 RNA extraction and analysis by real-time qPCR Destination: s2_3 Item: Title: 2.4 Protein extraction, SDS-PAGE, and In-Gel detection of NanoLuc Destination: s2_4 Item: Title: 2.5 Determination of NanoLuc activity Destination: s2_5 Item: Title: 2.6 Determination of Luz activity Destination: s2_6 Item: Title: 2.7 Tryptamine extraction and quantification Destination: s2_7 Item: Title: 3 Results Destination: s3 Children: Item: Title: 3.1 An intron insertion approach that adds flexibility to transgene assemblies Destination: s3_1 Item: Title: 3.2 Level 0 parts are easily assembled using MoInClo Destination: s3_2 Item: Title: 3.3 Assembly into functional transcriptional units driving NanoLuc expression Destination: s3_3 Item: Title: 3.4 Expression of a fungal luciferase implements part of a bioluminescence pathway in C. reinhardtii Destination: s3_4 Item: Title: 3.5 Overexpression of a tryptophan decarboxylase does not result in tryptamine accumulation Destination: s3_5 Item: Title: 4 Discussion Destination: s4 Item: Title: Data availability statement Destination: bmark_-1258588262 Item: Title: Author contributions Destination: bmark_-1208584890 Item: Title: Funding Destination: bmark_1865464027 Item: Title: Acknowledgments Destination: bmark_-366698314 Item: Title: Conflict of interest Destination: bmark_1089982005 Item: Title: Generative AI statement Destination: 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Front. Plant Sci. 16:1544873. doi: 10.3389/fpls.2025.1544873 Copyright © 2025 Aschern, Braad, Milito, Alzuria and Yang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. synthetic biology Chlamydomonas reinhardtii transgene expression intron-mediated enhancement (IME) Modular Cloning (MoClo) microalgal biotechnology A novel MoClo-mediated intron insertion system facilitates enhanced transgene expression in Chlamydomonas reinhardtii synthetic biology; Chlamydomonas reinhardtii; transgene expression; intron-mediated enhancement (IME); Modular Cloning (MoClo); microalgal biotechnology True https://www.frontiersin.org/legal/copyright-statement Pages: Page: Label: 1 Annotations: Annotation: Subtype: Link Rect: 221, 685, 529, 706 Annotation: Subtype: Link Rect: 221, 660, 477, 681 Annotation: Subtype: Link Rect: 221, 635, 528, 656 Annotation: Subtype: Link Rect: 221, 610, 512, 631 Annotation: Subtype: Link Rect: 381, 144, 436, 153 Destination: 208 Annotation: Subtype: Link Rect: 303, 69, 414, 78 Destination: 208 Annotation: Subtype: Link Rect: 419, 69, 483, 78 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