A MARS1 ‘midigene’ was generated by amplifying four different portions of this gene either from gDNA or cDNA using KOD Hot Start DNA Polymerase (Thermo Fisher Scientific) or Phusion Hotstart II polymerase (Thermo Fisher Scientific). In particular, the region spanning the promoter, the 5’UTR and the first 5 exons of this gene was amplified from gDNA using Phusion polymerase and the following primers: SR828 and SR818; the region spanning exon 5 to exon 15 was amplified from gDNA using KOD polymerase and the following primers: SR819 and HT7; the region spanning exon 15 to exon 28 was amplified from cDNA using KOD polymerase and the following primers: SR789 and SR797 and the 3’UTR was amplified from gDNA using KOD polymerase and the following primers: SR793 and SR829.
All PCR products were gel extracted and purified as described above in the section regarding the pPW3217 cloning. Next, these 4 PCR fragments were mixed with a purified and linearized and pRAM118/pPW3216 vector, previously digested by EcoRV and Not1, and incubated in presence of the In-Fusion reagents (Takara) as per manufacturer’s instructions. The resulting plasmid is notated as pPW3218. The sequence of the aforementioned primers can be found inTable 3 .
The Phytozome v5.5 MARS1 transcript annotation is Cre16.g692228.t1.1.
All PCR products were gel extracted and purified as described above in the section regarding the pPW3217 cloning. Next, these 4 PCR fragments were mixed with a purified and linearized and pRAM118/pPW3216 vector, previously digested by EcoRV and Not1, and incubated in presence of the In-Fusion reagents (Takara) as per manufacturer’s instructions. The resulting plasmid is notated as pPW3218. The sequence of the aforementioned primers can be found in
The Phytozome v5.5 MARS1 transcript annotation is Cre16.g692228.t1.1.