Fig. 2.

Strategy for the assembly and cloning of the antigenic gene into mRNA backbone plasmid. The DNA encoding the antigenic protein was designed based on the metadata of B.1.1.529 infected humans. The unique restriction sites at the 5′ and 3′ end of the coding gene and a few unique sites were incorporated in-between regions to facilitate the cloning. A total of 158 oligonucleotides that included 81 forward and 77 reverse oligonucleotides were designed and used for the assembly of the entire DNA. For the PCR-based assembly of the final DNA, fragments A, B, C, and D were annotated, and they were flanked by unique restriction sites. Fragment A was assembled using sub-fragments A1, and A2. Fragment B was assembled using sub-fragments B1, and B2. Fragment C1–C2 was assembled using sub-fragments C1, and C2. Fragment C3–C4 was assembled using sub-fragments C3, and C4. Fragment C was finally assembled using sub-fragments C1–C2, and C3–C4. Fragment D was assembled using sub-fragments D1, and D2. Finally, AB was assembled using A and B, and similarly, CD using C and D fragments. For the cloning of the synthesized fragment AB and CD into the pVEE-104a.1 plasmid backbone, each was digested with their respective restriction enzyme cutters and finally ligated by three-fragment ligation to create pVEE-101c.1. Oligonucleotides used here are given the “ML” prefix followed by the numerical numbers. The oligonucleotides mentioned in the underlined text are the forward and those which are in italics are the reverse oligonucleotides. The single pair of primers that are used for the final assembly is mentioned for each of the sub-fragments and fragments. The unique restriction sites are also mentioned flanking the fragments.