How can I alter the reading frame when performing In-Fusion Cloning?
How can I alter the reading frame when performing In-Fusion Cloning?
The reading frame is defined by the primer sequence. For example, when creating a fusion protein, if the 15 bp of vector homology at the 5' end of the In?Fusion PCR primer sequence corresponds to the last five codons of the vector reading frame, you would clone your new gene or tag in the same reading frame downstream of the C-terminus of the vector sequence by placing the first codon of this gene next to the last codon of the homology sequence (i.e., at the 3' end) without any interfering STOP codons. To shift the reading frame, you would simply add one or two additional bases after the 15-bp homology and before the first codon of the target gene.