Introduction <p>Optimized vectors are crucial in modern molecular biology because they greatly enhance the efficiency of the DNA cloning procedure. This study aimed to design a novel engineered vector containing an HphI cassette to enhance TA cloning efficiency.</p> Methods <p>The pTZ57R/T plasmid served as the basis for developing the novel Engineered vector (pTZ57R/T-2000). Three restriction sites (respectively, XbaI- HphI -SalI) were inserted into amplified fragments by specific primers. The PCR amplified fragments (1,000 bp) were digested by XbaI and SalI enzymes, then ligated into the modified pTZ57R/T plasmid (pTZ57R/T-HphI), which had been prepared by digestion with the same enzymes. Finally, the transformation and cloning efficiencies of the PCR products were assessed through the number of clones and colony PCR, respectively.</p> Results <p>The statistical analyses showed a significant difference in transformation efficiency between pTZ57R/T-2000 and ddT-tailed vectors (P &lt; 0.0001), with the pTZ57R/T-2000 vector producing an average of 54 clones compared to 26.67 clones for the ddT-tailed vectors. However, there was no significant difference in TA cloning efficiency between the two vectors (P = 0.1069).</p> Discussion <p>The success rate of pTZ57R/T-2000 vector transformation is higher than ddT-tailed vector in terms of the number of clones. However, the TA cloning efficiency is lower in pTZ57R/T-2000 vectors compared to ddT-tailed vectors, to eliminate the assumption of a shortage of genes for cloning. To enhance the pTZ57R/T-2000 efficiency, it is recommended to use pre-divided tubes to reduce T-head damage during defreezing and pipetting.</p> Graphical Abstract <p></p>