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Background <p>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.</p> Results <p>Here, we engineered an insertion-activated cloning vector that accepts PCR products directly. In this study, we verified that 60-bp <i>NptII</i> promoters can effectively drive the expression of the <i>mScarlet-I</i> gene. In contrast, the 45-bp <i>NptII</i> promoter can’t drive the expression of the <i>mScarlet-I</i> gene. The insertion-activated cloning vector harbored with the 45-bp truncated <i>NptII</i> promoter and ORF of <i>mScarlet-I</i> gene. This cloning vector contains an <i>Eco</i>RⅤ 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 <i>NptII</i> 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 <i>NptII</i> promoter can be connected and drives expression of the reporter gene, <i>mScarlet-I</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.</p> Conclusions <p>The positive selection marker in this insertion-activated cloning vector can achieve higher screening efficiency.</p>

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Construction and application of an insertional activation cloning vector

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月19日

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  • Yan Zhang,
  • Yun He,
  • Yi Ding,
  • Shanhua Lyu,
  • Yinglun Fan

摘要

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.