Minigene construction and expression
The minigene expression plasmids were constructed as previously reported
(Beck et al., 2013) and in vitro splicing confirmation was
performed follow before (Volodarsky et al., 2015). pSPL3, a cloning
expression plasmid, was purchased from Fenghui Biotechnology (China).
Briefly, the vector was cleaved with Bam HI (Takara Bio, USA) andEco RI (Takara Bio), and then ligated the DNA segment containing
the two mutation sites to the pSPL3 vector using T4 ligase (NEB, USA)
following the manufacturer’s instruction. The primers were as follows:
dup-F: 5’-CGCTCGAGTTTGTTTACTG TCCTATCTTAAGCAC-3’, dup-R:
5’-CGGGATCCACCTGATTAGGAGGATTGTTGC-3’, 82-2-F:
5’-CGCTCGAGAAGCCAATGTCTAACAGATAAT-3’, 82-2-R: 5’-CGGGATCCCTTGTTTCCCCCATT
CTT-3’.