The plasmids pcDNA-FLAG-RBM4(a) and pEGFP-CAD (encoding residues 196–364 of RBM4a) have been described (2 (link)). Using a polymerase chain reaction (PCR)-based gene deletion strategy, Ala-tract #1 (residues 233–239) and #2 (residues 282–299) of pcDNA-FLAG-RBM4(a) were removed to generate pFLAG-RBM4-A0. The pFLAG-RBM4-A25 plasmid was constructed by substituting three nonalanine residues of Ala-tract #2 with alanines and inserting an additional seven alanines. The RBM4b-coding region was cloned into pcDNA3.1 in frame with the FLAG sequence. The coding regions of CAD-A0, CAD-A25, PSF and SRSF1/ASF were each inserted into vector pEGFP-C1 (Invitrogen) to generate the respective C-terminal green fluorescent protein (GFP) fusions. The pcDNA-FLAG-CoAZ was a kind gift of Lan Ko (Medical College of Georgia). The red fluorescent protein (RFP)-coding sequence was inserted into pcDNA-FLAG-RBM4(a) and pcDNA-FLAG-CoAZ to generate vectors RFP-RBM4(a) and RFP-CoAZ, respectively. The splicing reporter plasmids have been described (3 (link)).
Protocol detail
Plasmid Construction for RBM4 and CAD Variants
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Alanines
Coding sequence
Frame
Gene deletion
Green fluorescent protein
Plasmids
Polymerase chain reaction
Red fluorescent protein
Vectors
Corresponding Organization : Academia Sinica
Other organizations : Institute of Molecular Biology, Academia Sinica
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Variable analysis
independent variables
- Removal of Ala-tract #1 (residues 233-239) and #2 (residues 282-299) of pcDNA-FLAG-RBM4(a) to generate pFLAG-RBM4-A0
- Substitution of three nonalanine residues of Ala-tract #2 with alanines and insertion of an additional seven alanines to generate pFLAG-RBM4-A25
- Measurement of the effects of the Ala-tract modifications on the RBM4a and RBM4b proteins
- The plasmids pcDNA-FLAG-RBM4(a) and pEGFP-CAD (encoding residues 196–364 of RBM4a)
- The RBM4b-coding region cloned into pcDNA3.1 in frame with the FLAG sequence
- The coding regions of CAD-A0, CAD-A25, PSF and SRSF1/ASF each inserted into vector pEGFP-C1 to generate the respective C-terminal green fluorescent protein (GFP) fusions
- The pcDNA-FLAG-CoAZ plasmid
- The RFP-RBM4(a) and RFP-CoAZ vectors
- The splicing reporter plasmids
- The plasmids pcDNA-FLAG-RBM4(a) and pEGFP-CAD (encoding residues 196–364 of RBM4a)
- No negative controls were explicitly mentioned in the provided information.
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