<p>The plasmid construct consists of 8 fragments, which include a promoter, six genes with their corresponding RBS sequences, and a terminator. The total length of the insert, excluding the plasmid backbone, is 10,168 base pairs. In order to facilitate synthesis and assembly, the fragments have been divided into four gBlocks of approximately 3,000 bp each. Each gBlock has been designed with overlapping sequences (overhangs) required for Gibson assembly to ensure precise and efficient integration of the fragments into the pSEVA261 vector backbone. Prior to assembly simulation in SnapGene, the sequences of all fragments were codon-optimised with Benchling's codon optimisation tool and verified with a personal codon frequency table obtained from (Source) to enhance translational efficiency and allow for proper gene expression in V. natriegens.</p>
<p>For linearisation of the pSEVA261 plasmid, PCR primers have been designed. These primers contain sequences that are complementary to the overhangs of Fragment 1 and Fragment 4. Notably, the primers also exclude the multiple cloning site, which is in turn replaced by the fragments.</p>
<p>Each fragment has a corresponding ribosome binding site (RBS) positioned immediately upstream of the start codon to ensure proper translation. The native P1 promoter is located upstream of the gene fragments to initiate transcription. The terminator is introduced downstream of the gene sequences for proper transcription termination. Since the gBlocks have been divided into four segments, an additional set of eight primers have been designed with sequences complementary to adjacent fragments, ensuring proper alignment for Gibson assembly. Each primer adds approximately 40 base pairs of overlap, following guidelines from SnapGene, to facilitate seamless assembly of the fragments. The final recombinant plasmid has a total length of 12,413 base pairs.</p>
