【Abstract】 Objective To construct a lentivirus based library containing a fused promoter of 20N random sequence plus miniCMV, which drives the puromycin resistant gene PAC expression, for screening of response elements under specific contexts.Methods Primers specific for puromycin resistant gene PAC was designed and the amplicons were achieved via PCR. Then the amplicons were cloned into the lentivirus vector pWPI with the help of restriction enzyme Pac I and Pme I. An adaptor sequence plus 20N random sequence together with miniCMV sequence were designed and synthesized. The complementary strand of the synthesized sequence was achieved by PCR reaction with the help of a primer. The obtained double strand DNA were then cloned into the above pWPIPAC vector, generating the library. The library was transfected into Hela cells and the cells were subjected to ultrasound irradiation, with the parameter of 100mW/cm2 for 10 minutes each day and continued for 3 days. The processed cells were additionally treated with puromycin and the DNA in the survived cells at day 4 were isolated and the exact sequence of the 20N in these cells were identified by sequencing.Results We here proposed a strategy to construct random promoters driving selection gene expression. The constructed reporter library contained 20N, miniCMV and PAC gene. Under ultrasound irradiation, these cloned containing promoters upregulated by ultrasound drives the PAC gene expression and thus more resistant to puromycin induced cell death. The detailed information of these promoters were able to be characterized by sequencing the 20N in the survived cells by sequencing the amplicons corresponding to the 20 N.Conclusion We have successfully constructed a lentivirus based random library, in which puromycin resistant gene PAC was driven by random fused promoters. Together with puromycin treatment, the library can be applied for identification of specific ciselements under certain contexts. What’s more, these identified ciselements could be promising in controlling spatiotemporal gene expression.