To research the effects of vector quantity and competence on the positive cloning rate, with a known gene sequence but in the absence of DNA template, we artificially designed 26 primers to synthesize a target gene of 835 bp in vitro using overlapping PCR technique. The whole experiment design with two factors and six levels (36 combinations) was applied to study the effects of the vector density and competent cells on the macromolecular vector transformation efficiency. Based on the 1 500 ng target gene, the vector density grades were designed (50, 100, 150, 200, 250, 300 ng), and then the recombinant plasmids were transformed into Top10F', DH5, Stbl3, Epi400, JM108, SCSI. Results showed that the positive cloning rates of different vector amount from big to small were in the order of 200,250,300,150,100 and 50 ng. The maximum positive cloning rate of 200 ng reached 75%; and the average value was 28. 5%. The positive cloning rates of different competent cells from big to small were in the order of stbl3, Top10F', DH5, JM108, Epi400 and SCSI. Stbl3 was higher than other competent cells under any vector density, and its average positive cloning rate was 42.4%. Both the vector density and competent cells had significant effects on the macromolecular vector transformation efficiency. The optimal combination was C4 with 200 ng vector density and Stbl3, the positive cloning rate of which could reach 75%.