SummarySynthetic small RNAs (sRNAs) are gaining increasing attention in the field of synthetic biology and bioengineering for efficient post-transcriptional regulation of gene expression. However, the optimal design of synthetic sRNAs is challenging because alterations may impair functions or off-target effects can arise. Here, we introduce DIGGER-Bac, a toolbox forDesign andIdentification of seed regions forGoldenGate assembly andExpression of synthetic sRNAs inBacteria. The SEEDling tool predicts optimal sRNA seed regions in combination with user-defined sRNA scaffolds for efficient regulation of specified mRNA targets. Results are passed on to the G-GArden tool, which assists with primer design for high-fidelity Golden Gate assembly of the desired synthetic sRNA constructs.