Here, an efficient cloning strategy for large DNA fragments and for simultaneous assembly of multiple DNA fragments assembly is presented. This strategy is named OEPR (based on Overlap Extension PCR and Recombination in vivo). OEPR cloning is a seamless, restriction- and ligation-independent method. The method takes advantage of both homologous recombination enzymes in E. coli and overlap PCR. ...

the ple2scx vector was developed for the stable expression of exogenous genes in the protozoan parasite leishmania. the ple2scx construct contains three independent selection markers: herpes simplex virus thymidine kinase (hsv-tk), cytosine deaminase (cd) and streptothericin acetyltransferase gene (sat) in multiple cloning site, flanking by 5’ and 3’ untranslated regions of the previously clone...

BACKGROUND Expression of higher eukaryotic genes as soluble, stable recombinant proteins is still a bottleneck step in biochemical and structural studies of novel proteins today. Correct identification of stable domains/fragments within the open reading frame (ORF), combined with proper cloning strategies, can greatly enhance the success rate when higher eukaryotic proteins are expressed as the...

Background and Aims: DNA cloning, sub-cloning and site directed mutagenesis are the most common strategies in nearly all projects of recombinant protein production. The classical method of restriction site cloning is unsatisfactory due to the need for supply of restriction enzymes and the inefficiency of the digestion reaction. Many new methods, including recombinatorial cloning and ligation in...

The pLE2SCX vector was developed for the stable expression of exogenous genes in the protozoan parasite Leishmania. The pLE2SCX construct contains three independent selection markers: herpes simplex virus thymidine kinase (HSV-TK), cytosine deaminase (CD) and streptothericin acetyltransferase gene (sat) in multiple cloning site, flanking by 5’ and 3’ untranslated regions of the previously clone...

Herein, we describe a novel cloning strategy for PCR-amplified DNA which employs the type IIs restriction endonuclease BsaI to create a linearized vector with four base-long 5'-overhangs, and T4 DNA polymerase treatment of the insert in presence of a single dNTP to create vector-compatible four base-long overhangs. Notably, the insert preparation does not require any restriction enzyme treatmen...

Background Eucalypt canker disease (Chrysoporthe cubensis) was the main driver of the concept of eucalypt clonal forestry in Brazil. Initially clonal forestry was based on outstanding spontaneous hybrids resistant to canker disease. Since the recognition that cloning superior inter-specific hybrids could be an important general strategy, several crosses were made for many different purposes, es...