COBALT is a constraint-based alignment tool for multiple protein sequences that improves alignment quality by incorporating constraints from databases like CDD and PROSITE. It uses a progressive alignment approach, combining pairwise constraints derived from sequence similarity, database searches, and user input. COBALT's performance is comparable to or better than existing tools like ClustalW, MUSCLE, ProbCons, and PCMA in terms of alignment accuracy and runtime. It is included in the NCBI C++ toolkit and available for download. COBALT's framework allows for the integration of biological information, such as conserved domains and motifs, to enhance alignment quality. It also allows users to specify regions of interest for alignment. COBALT uses RPS-BLAST to search for domains in CDD and aligns sequences based on these domain matches. It also incorporates PROSITE patterns for constraint generation, though these are less effective in initial stages. COBALT's performance is evaluated on various benchmarks, showing high alignment quality and efficiency. It is expected to be integrated into NCBI resources and further optimized for speed and accuracy. COBALT represents a good balance between alignment quality and runtime, making it a useful tool for multiple sequence alignment.COBALT is a constraint-based alignment tool for multiple protein sequences that improves alignment quality by incorporating constraints from databases like CDD and PROSITE. It uses a progressive alignment approach, combining pairwise constraints derived from sequence similarity, database searches, and user input. COBALT's performance is comparable to or better than existing tools like ClustalW, MUSCLE, ProbCons, and PCMA in terms of alignment accuracy and runtime. It is included in the NCBI C++ toolkit and available for download. COBALT's framework allows for the integration of biological information, such as conserved domains and motifs, to enhance alignment quality. It also allows users to specify regions of interest for alignment. COBALT uses RPS-BLAST to search for domains in CDD and aligns sequences based on these domain matches. It also incorporates PROSITE patterns for constraint generation, though these are less effective in initial stages. COBALT's performance is evaluated on various benchmarks, showing high alignment quality and efficiency. It is expected to be integrated into NCBI resources and further optimized for speed and accuracy. COBALT represents a good balance between alignment quality and runtime, making it a useful tool for multiple sequence alignment.