Abstract: Objective To explore the method for in vitro gene assembly of apoptin-encoding DNA sequence, for instance, using a large number of oligodeoxyribonucleotides(oligos). Methods Based on the encoding sequence of apoptin gene(GeneBank accession number AY171617), a number of oligos were designed to assembly apoptin gene in pfu mix reaction system, and each oligo was 40 nucleotides(nt) in length, in which synonymous codon substitution was used to eliminate the restriction enzyme sites of BglII( position 172, agatct→agatcc) and HindIII(position 306, aagctt→aatcct). The assembly mixture was further diluted and amplified with two end oligos. The targeting sequence was gel-purified, amplified for one more time, followed by the addition of T to the 3’ end in the presence of Taq polymerase and dATP before cloning into pGEM-T easy vector. The positive clones were confirmed by restriction enzyme digestion and sequence analysis. Results The synthetic mixture presented obvious "tails" in the first PCR for assembly. After dilution of the mixture and amplification with two end oligos, clear DNA ladder bands with a clear targeted band were yielded. After PCR, the targeted gene was cloned into pGEM-T easy vector, and the positive clones were confirmed by sequence analysis with be identical to the designed coding sequence of apoptin gene. Conclusion Gene assembly is a rapid and cost-effective approach for synthesis of genes or vectors, which allows simultaneous mutagenesis of several genes in vitro.