Abstract: The objective of this study was to solve the problem of construction a transgenic safety and site-specific integrated and marker-free LacS gene expression vector.Therefore,this experiment using plasmid pEGFP-N1 as the original framework,using PCR and restriction sites to add two with LoxP sequence at both ends of the marker genes in the pEGFP-N1 plasmid,attB sequence was added at the upstream of the multiple cloning sites for site-specific integration,then add the gene BC promoter-LacS-PolyA into the multiple cloning site.By restricted endonucleases digestion,these 3 fragments,Loxp,attB and LacS,were detected in the vector pEGFP-N1-LacS,PCR products and fragment size consistent with the expected results,the results of sequencing and oligonucleotide sequences also corresponding favorable,proved that each gene fragment was correctly connected to the corresponding position of the plasmid.In conclusion,the site-specific integrated and marker-free LacS gene vector was constructed successfully.

Key words: site-specific integrated; marker-free; LacS; pEGFP-N1 vector