A fixed gene copy number is important for the in silico construction of engineered synthetic networks. However, the copy number of integrated genes depends on their genomic location. This gene dosage effect is rarely addressed in synthetic biology. Two studies in Escherichia coli presented conflicting data on the impact of gene dosage. Here, we investigate how genome location and gene orientation influences expression in Bacillus subtilis. An important difference with the E. coli studies is that we used an unbiased genome integration approach mediated by random transposon insertion. We found that there is a strong gene dosage effect in fast growing B. subtilis cells, which can amount to a 5-fold difference in gene expression. In contrast, gene orientation with respect to DNA replication direction does not influence gene expression. Our study shows that gene dosage should be taken into account when designing synthetic circuits in B. subtilis and presumably other bacteria.