1887

Abstract

is the most prevalent fungal pathogen of humans. The current techniques used to construct strains require integration of exogenous DNA at ectopic locations, which can exert position effects on gene expression that can confound the interpretation of data from critical experiments such as virulence assays. We have identified a large intergenic region, , which facilitates the integration and expression of ectopic genes. To construct and integrate inserts into this novel locus, we re-engineered yeast/bacterial shuttle vectors by incorporating 550 bp of homology to . These vectors allow rapid, facile cloning through recombination (gap repair) in and efficient integration of the construct into the locus. Other useful features of these vectors include a choice of three selectable markers (, the recyclable or ), and rare restriction enzyme recognition sites for releasing the insert from the vector prior to transformation into , thereby reducing the insert size and preventing integration of non- DNA. Importantly, unlike the commonly used locus, integration at has no negative effect on growth rates and allows native-locus expression levels, making it an ideal genomic locus for the integration of exogenous DNA in .

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2013-03-01
2019-08-25
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