The ɸC31 integrase system is widely used in Drosophila melanogaster to allow transgene targeting to specific loci. Over the years, flies bearing any of more than 100 attP docking sites have been constructed. One popular docking site, termed attP40, is located close to the Nesprin-1 orthologue msp-300 and lies upstream of certain msp-300 isoforms and within the first intron of others. Here we show that attP40 causes larval muscle nuclear clustering, which is a phenotype also conferred by msp-300 mutations. We also show that flies bearing insertions within attP40 can exhibit decreased msp-300 transcript levels in third instar larvae. Finally, chromosomes carrying certain “transgenic RNAi project” (TRiP) insertions into attP40 can confer pupal or adult inviability or infertility, or dominant nuclear clustering effects in certain genetic backgrounds. These phenotypes do not require transcription from the insertions within attP40. These results demonstrate that attP40 and insertion derivatives act as msp-300 insertional mutations. These findings should be considered when interpreting data from attP40-bearing flies.