Engineering BRCA1 and RAD18 TULIP2 Constructs

BRCA1 gene was amplified and STOP codon removed from pDEST-FRT/T0-GFP-BRCA1 (Addgene #71116) by PCR using BP-tailed primers and cloned into pDNOR207 by Gateway® cloning BP reaction (Thermo Fisher Scientific). BRCA1-I26A mutation was introduced by site-directed mutagenesis. RAD18-pDNOR223 plasmid was obtained from Open Biosystems (Clone 1782) and the STOP codon was removed by site-directed mutagenesis. TULIP2 constructs were generated by Gateway® cloning LR reaction between donor plasmids containing BARD1^{28 (link)}, BRCA1 or RAD18 and acceptor TULIP2 plasmids^{27 (link)}. For the GFP-tagged constructs, the LR reactions were performed with pLVU-GFP, gift from Lars Ittner (Addgene #24177)^{59 (link)}. Primers are listed in Supplementary Table 1.

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Salas-Lloret D., García-Rodríguez N., Soto-Hidalgo E., González-Vinceiro L., Espejo-Serrano C., Giebel L., Mateos-Martín M.L., de Ru A.H., van Veelen P.A., Huertas P., Vertegaal A.C, & González-Prieto R. (2024). BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis. Nature Communications, 15, 4292.

Publication 2024

pDEST-FRT/T0-GFP-BRCA1 Gateway® cloning BP reaction

Corresponding Organization :

Other organizations : Leiden University Medical Center, Centro Andaluz de Biología Molecular y Medicina Regenerativa, Universidad Pablo de Olavide, Junta de Andalucía, Universidad de Sevilla, Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío, Netherlands Metabolomics Centre

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Variable analysis

independent variables

BRCA1 gene amplification and STOP codon removal
BRCA1-I26A mutation introduction
RAD18 STOP codon removal
TULIP2 constructs generation by Gateway® cloning LR reaction

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

controls

Positive control: None mentioned
Negative control: None mentioned

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