Efficient Plasmid Construction via USER Cloning and CRISPR/Cas9

USER cloning^{37 (link)} was used to construct plasmids, unless otherwise specified, and the EasyClone MarkerFree system with integration plasmids and gRNA plasmids compatible with CRISPR/Cas9^{38 (link)} were used throughout. USER-compatible vectors were treated with FastDigest SfaAI (Thermo Fisher Scientific) and Nb.BsmI (NEB) prior to ligation, and USER-compatible fragments were amplified with Phusion U Hot Start PCR Master Mix (Thermo Fisher Scientific) to read through uracil overhangs contained in oligos. Strains, plasmids, oligos, gene blocks, and heterologous GPCR accession IDs are listed in Supplementary Data 4–8.

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Jensen E.D., Deichmann M., Ma X., Vilandt R.U., Schiesaro G., Rojek M.B., Lengger B., Eliasson L., Vento J.M., Durmusoglu D., Hovmand S.P., Al’Abri I., Zhang J., Crook N, & Jensen M.K. (2022). Engineered cell differentiation and sexual reproduction in probiotic and mating yeasts. Nature Communications, 13, 6201.

Publication 2022

FastDigest SfaAI Nb.BsmI Phusion U Hot Start PCR Master Mix

Crispr Gene Ligation Oligos Plasmids Strains Uracil Vectors

Corresponding Organization : Technical University of Denmark

Other organizations : North Carolina State University

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Variable analysis

independent variables

USER cloning method used to construct plasmids
EasyClone MarkerFree system with integration plasmids and gRNA plasmids compatible with CRISPR/Cas9

dependent variables

Not explicitly mentioned

control variables

Strains, plasmids, oligos, gene blocks, and heterologous GPCR accession IDs listed in Supplementary Data 4–8

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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