Cloning and Expression of Protein Constructs

3xFLAG-tagged SANS full length and deletion constructs were designed and cloned in house as previously described (29 (link)). HA-PRPF31 constructs were kind gifts from Dr. Utz Fischer (41 (link)). Deletion constructs of PRPF31 (N-term (aa1–165), C-term (aa 166–499)) were produced by PCR from the HA-PRPF31 plasmid and cloned into the pcDNA3.1 N-HA vector (Thermo Fisher). Deletion constructs of PRPF6 (NTD (aa 1–126), HAT1-6 (aa 293–592), HAT7-13 (aa 593–941)) were cloned into AcGFP-C1 vector (Addgene: 64607). RON (MST1R) minigene was a kind gift from Dr. Julian Koenig (IMB-Mainz), and USH1C minigene was amplified from the genomic DNA of HEK293T cells and cloned into the pcDNA5/FRT vector (Thermo Fisher). The pcDNA5/FRT-PRPF4 plasmid was constructed by cloning the PRPF4 gene fused to an N-terminal FLAG/HA tag into the pcDNA5/FRT plasmid (Thermo Fisher).

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Yildirim A., Mozaffari-Jovin S., Wallisch A.K., Schäfer J., Ludwig S.E., Urlaub H., Lührmann R, & Wolfrum U. (2021). SANS (USH1G) regulates pre-mRNA splicing by mediating the intra-nuclear transfer of tri-snRNP complexes. Nucleic Acids Research, 49(10), 5845-5866.

Publication 2021

pcDNA3.1 N-HA vector AcGFP-C1 vector pcDNA5/FRT vector pcDNA5/FRT plasmid

Cells Deletion Gene Genomic Gifts Hat1 Plasmid Ush1c Vector

Corresponding Organization :

Other organizations : Johannes Gutenberg University Mainz, Max Planck Institute for Biophysical Chemistry, Mashhad University of Medical Sciences, Reference Institute for Bioanalytics

Top 5 similar protocols

Variable analysis

independent variables

3xFLAG-tagged SANS full length and deletion constructs
HA-PRPF31 constructs
Deletion constructs of PRPF31 (N-term (aa1–165), C-term (aa 166–499))
Deletion constructs of PRPF6 (NTD (aa 1–126), HAT1-6 (aa 293–592), HAT7-13 (aa 593–941))
RON (MST1R) minigene
USH1C minigene
PcDNA5/FRT-PRPF4 plasmid

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

positive controls

None specified

negative controls

None specified

Annotations

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