Abstract: A detection of optimal genetic recombinants used to prepare target proteins, with assessment of their target-specific “upstream” productivity, genetic stability and means to optimize target protein “downstream” purification using customizable fluorescent tags. A scarless removable protein fusion makes it possible to identify recombinants of Pichia pastoris with optimal performance in heterologous protein production.

Abstract: A method for assembling polynucleic acids that introduces phosphorothioate bonds into the linker oligos during the assembly procedure in order to use exonucleases to isolate the DNA of interest, thereby eliminating any cumbersome purification steps, such as gel electrophoresis and significantly increasing the overall selectivity and efficiency of the method. The present invention introduces a phosphorothioate bond by replacing a non-bridging oxygen within the phosphate backbone of the nucleic acid sequence with a sulfur (S) atom. Introduction of this sulfur atom results in an internucleotide linkage that is resistant to nuclease cleavage. Consequently, by adding such modified S linkers to form nuclease resistant ends of part-linker DNA, the present invention allows the use of exonucleases to degrade parts that do not have linkers ligated to their ends to isolate only the part-linker DNA of interest to increase assembly efficiency and selectivity and avoiding the need for purification by gel electrophoresis.