Difference between revisions of "Part:BBa K5142045"

Latest revision as of 12:11, 2 October 2024

Compositive construct for A27L-3Stop mutagenesis

This part was used to replace the wild-type A27L coding sequence in vaccinia virus (VACV) genome with A27L-3Stop, via homologous recombination in VACV genome. This part includes four functional elements in sequence: 1) vaccinia virus A28L, which serves as the left homologous arm; 2) GFP cds regulated by poxviral H5 promoter in reverse direction, which is used to indicate the success of VACV infection and proximity of the construct to viral genome; 3) A27L-3Stop regulated by overlapping early/late poxviral promoter, which is the core sequence for recombination; 4) partial sequence of A26L, which serves as the right homologous arm. The structure of this construct is shown in the following figure.

Figure 1.Schematic diagram showing the structure of the compositive construct for A27L-3Stop mutagenesis. The arrows indicate the directions of transcription and the BioBrick scars are omitted.

This part is composed of the following basic parts aligned tandemly: vaccinia virus A28L (BBa_K5142010), reverse complementary GFP cds (BBa_K5142013), reverse poxviral H5 promoter (BBa_K5142012), overlapping early/late poxviral promoter (BBa_K5142011), vaccinia virus A27L-3stop (BBa_K5142007), vaccinia virus A26L (partial) (BBa_K5142014).

1. To prevent potential readthrough transcription when inserted into vaccinia virus genome, the GFP was transcribed in the direction reverse to the adjacent viral genes (e.g., A26L, A27L and A28L) which was driven by poxviral H5 promoter.
2. Considering the whole A26L sequence is too long as a homologous arm, we chose 500 bp sequence adjacent to A27L only to construct the recombinant element.