Construction and application of an insertional activation cloning vector

BACKGROUND: Foreign DNA cloning is a critical step in genetic engineering and molecular biology for acquiring target genes. Efficient and accurate identification of positive clones from transformants is essential to this process. RESULTS: Here, we engineered an insertion-activated cloning vector that accepts PCR products directly. In this study, we verified that 60-bp NptII promoters can effectively drive the expression of the mScarlet-I gene. In contrast, the 45-bp NptII promoter can’t drive the expression of the mScarlet-I gene. The insertion-activated cloning vector harbored with the 45-bp truncated NptII promoter and ORF of mScarlet-I gene. This cloning vector contains an EcoRⅤ restriction site, which serves as the cloning site for PCR products. When amplifying exogenous DNA, the designed reverse primer is added with the 15-bp core sequence from − 60 to -46 of the NptII promoter. If the PCR product is cloned into the insertion-activated cloning vector with the correct orientation, the 15-bp core promoter and the 45-bp truncated NptII promoter can be connected and drives expression of the reporter gene, mScarlet-I, which allows positive recombinants to be identified with red fluorescence. Five PCR products with different sizes were tested using the one-step restriction enzyme digestion and ligation approach or in vivo cloning method. The. CONCLUSIONS: The positive selection marker in this insertion-activated cloning vector can achieve higher screening efficiency.