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Enzymatic assembly of small synthetic genes with repetitive elements

View ORCID ProfileMichael T. A. Nguyen, Martin Vincent Gobry, Néstor Sampedro Vallina, Georgios Pothoulakis, View ORCID ProfileEbbe Sloth Andersen
doi: https://doi.org/10.1101/2022.11.23.517630
Michael T. A. Nguyen
1Interdisciplinary Nanoscience Center (iNANO), Gustav Wieds Vej 14, Aarhus University, DK-8000 Aarhus, Denmark
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  • For correspondence: michaelnguyen91@live.dk esa@inano.au.dk
Martin Vincent Gobry
1Interdisciplinary Nanoscience Center (iNANO), Gustav Wieds Vej 14, Aarhus University, DK-8000 Aarhus, Denmark
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Néstor Sampedro Vallina
1Interdisciplinary Nanoscience Center (iNANO), Gustav Wieds Vej 14, Aarhus University, DK-8000 Aarhus, Denmark
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Georgios Pothoulakis
1Interdisciplinary Nanoscience Center (iNANO), Gustav Wieds Vej 14, Aarhus University, DK-8000 Aarhus, Denmark
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Ebbe Sloth Andersen
1Interdisciplinary Nanoscience Center (iNANO), Gustav Wieds Vej 14, Aarhus University, DK-8000 Aarhus, Denmark
2Department of Molecular Biology and Genetics, Gustav Wieds Vej 14, Aarhus University, DK-8000 Aarhus, Denmark
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  • For correspondence: michaelnguyen91@live.dk esa@inano.au.dk
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Abstract

Although gene synthesis efficiency has improved through the years, current methods offered by DNA synthesis vendors are limited when it comes to repetitive sequences. Here, we describe a method for the enzymatic assembly of repetitive small synthetic genes. The method involves an initial step where the gene of interest is split in silico into small synthons of up to 80 base pairs flanked by Golden Gate-compatible four-base pair overhangs. Synthons are enzymatically synthesized by oligo extension and then assembled into the synthetic gene by Golden Gate Assembly. We construct eight different synthetic genes ranging from 133 to 456 base pairs encoding RNA structures with repetitive elements that are challenging to synthesize. We report assembly fidelities of up to 87.5 % that decrease for increasing number of synthons. This method is envisioned as an important addition to the molecular cloning toolbox and especially useful for construction of challenging and repetitive genes.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Competing Interest Statement: No competing interests.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted November 23, 2022.
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Enzymatic assembly of small synthetic genes with repetitive elements
Michael T. A. Nguyen, Martin Vincent Gobry, Néstor Sampedro Vallina, Georgios Pothoulakis, Ebbe Sloth Andersen
bioRxiv 2022.11.23.517630; doi: https://doi.org/10.1101/2022.11.23.517630
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Enzymatic assembly of small synthetic genes with repetitive elements
Michael T. A. Nguyen, Martin Vincent Gobry, Néstor Sampedro Vallina, Georgios Pothoulakis, Ebbe Sloth Andersen
bioRxiv 2022.11.23.517630; doi: https://doi.org/10.1101/2022.11.23.517630

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