Construction of a T-vector using an esterase reporter for direct cloning of PCR products.
We constructed an efficient T vector pTQEST216T that employed an engineered esterase as an indicator for direct cloning of PCR products. After ligation of the XcmI-digested vector with PCR products, this cloning system could easily discriminate positive clones due to insertional inactivation of the esterase reporter. Additionally, PCR products were cloned into this vector efficiently without the gel purification steps, due to the well-designed multi-cloning site that was in-frame fused at the circularly permutated gap of the reporter.
Department of Biological Sciences, College of Natural Sciences, Chonnam National University, Gwang-ju 500-757, Korea.
Ho-Dong
HD
Lim
Dae-Eun
DE
Cheong
Hyun-Jae
HJ
Shin
Geun-Joong
GJ
Kim
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004950:Esterases / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction; D015202:Protein Engineering
D004950:Esterases