Construction of two pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites for direct cloning of PCR products.
For applications such as sequencing, transfection, and in vitro transcription, PCR products have to be subcloned into plasmids. Many strategies are used for cloning, blunt-end ligation or the incorporation of restriction endonuclease sites into PCR primers for appropriate vectors. However, the most convenient and direct method is T/A cloning. In this study, we developed two of the pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites, and these T vectors have all the features of pGEM-7Zf(+): f1 ori, T7, and SP6 RNA polymerase promoters, the alpha-peptide coding region of beta-galactosidase for X-gal blue/white color selection, the beta-lactamase gene for recombinant colony selection, and binding sites for pUC/M13 forward and reverse sequencing primers. These AhdI-containing phagemid vectors, pGEM-NJ105 and pGEM-NJ107, are useful for the easy and inexpensive preparation of T vectors and direct cloning of PCR products.
Jeung
Ji Ung
JU
Graduate School of Biotechnology, Korea University, Seoul 136-701, South Korea.
Cho
Sung Ki
SK
Shim
Kyu Suk
KS
Ok
Sung Han
SH
Lim
Dae Sik
DS
Shin
Jeong Sheop
JS
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D014170:Transformation, Genetic
D004262:DNA Restriction Enzymes