Pre-mRNA splicing is an essential step in gene expression, when introns are removed and exons joined by the complex of proteins called spliceosome. Correct splicing requires a precise exon/intron junction definition, which is determined by a consensual donor and acceptor splice site at the 5 and 3 end, respectively. An acceptor splice site (3ss) consists of highly conserved AG nucleotides in positions E-2 and E-1. These nucleotides can appear in tandem, located 3bp from each other. Then they are referred to as NAGNAG or tandem 3ss, which can be alternatively spliced. NAG/TAG 3ss motif abundance is extremely low and cannot be easily explained by just a nucleotide preference in this position. We tested artificial NAG/TAG motif's potential negative effect on exon recognition using a minigene assay. Introducing the NAG/TAG motif into seven different exons revealed no general negative effect on exon recognition. The only observed effect was the partial use of the newly formed distal 3ss. We can conclude that this motif's extremely low preference in a natural 3ss is not a consequence of the NAG/TAG motif's negative effect on exon recognition, but more likely the result of other RNA processing aspects, such as an alternative 3ss choice, decreased 3ss strength, or incorporating an amber stop codon.