title: Construction of a restriction-endonuclease-Eam1105I-generated T-vector for high-throughput cloning and expression.
abstract: A novel T-vector was constructed that could be used for direct cloning and expression of PCR-amplified cDNA. The technique was based on the insertion into the parent vector of two endonuclease-Eam1105I restriction sequences spaced by an expression cassette of the full-length beta-galactosidase, which helped to improve cloning efficiency and to minimize the non-recombinant background of the T-vector when used to clone PCR products. Moreover, this method took advantage of the reconstitution of the rarest restriction sequence of MssI to enable directional cloning. These advantages make the T-vector suitable for high-throughput expression and analysis.
authors.affiliation: Key Research Laboratory of Hormone and Development Affiliated to the Ministry of Health, Institute of Endocrinology, Tianjin Medical University, Tianjin, People's Republic of China.
authors.forename: Baoli
authors.initials: B
authors.lastname: Wang
authors.forename: Hui
authors.initials: H
authors.lastname: Liang
authors.forename: Rui
authors.initials: R
authors.lastname: Liu
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Li
authors.forename: Bei
authors.initials: B
authors.lastname: Sun
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Shanyi
authors.initials: S
authors.lastname: Guo
authors.forename: Gang
authors.initials: G
authors.lastname: Guo
authors.forename: Jingyu
authors.initials: J
authors.lastname: Zhang
authors.forename: Chenlin
authors.initials: C
authors.lastname: Dai
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C075914:endodeoxyribonuclease Eam1105I; D015252:Deoxyribonucleases, Type II Site-Specific