Objectives: The primary objectives of this study were to develop an objective nucleic acid sequence screening framework and to leverage the framework for an empirical sensitivity study that measures the impact of ambiguities in regulatory and guidance documentation regarding the control of synthetic nucleic acids and screening of nucleic acid orders. Methods: Foundational risk levels were constructed using the bioinformatic sequencing screening tool UltraSEQ. The risk levels range from high (corresponding to regulated sequences) to low (corresponding to nonregulated sequences of concern) to no-risk. A representative sequence data set (141,651 sequences) was constructed from publicly available synthetically derived sequences, and the percentage sequences in each risk level was determined, followed by the impact of changing key UltraSEQ parameters. Results: The results of this study show that no-risk sequences represent 90-92% of sequences, and nonregulated sequences of concern represented 7-9% of the sequences regardless of the parameters. The parameter with the biggest impact on the number of sequences flagged was the minimum hit homology level, followed by minimum sequence region length, and finally uniqueness of the hit to a select agent sequence. Conclusion: The results of this empirical study provide a greater understanding for gene synthesis providers, biosafety and biosecurity practitioners, and the scientific community regarding the impact of various interpretations of regulatory and guidance documentation. The risk level framework provides a foundation to build upon for nucleic acid sequence screening as the threat landscape evolves. However, additional development is needed to build tools that connect predictions across sequences and orders to provide contextual risk-based predictions.