Provided herein are methods for assembling DNA fragments employing at least three enzymatic activities: DNA polymerase, flap endonuclease, and DNA ligase. Certain aspects include methods for generating closed circular DNA products, e.g., plasmid vectors, by assembling various DNA fragments having complementary ends that hybridize to one another. The resulting circular products can be introduced into host cells and selected for desired properties. Kits for performing the method are also provided.

A method for synthesizing a target double stranded (ds) polynucleotide byproviding an oligonucleotide library within an array device that has a diversity of oligonucleotide library members, each of which has a different nucleotide sequence and is contained in a separate library containment in an aqueous solution. The library includes single stranded oligonucleotides and double stranded oligonucleotides with at least one overhang and covers at least 10,000 pairs of matching oligonucleotides. In a first step, at least a first pair of matching oligonucleotides are transferred transferred from the library into a first reaction containment using a liquid handler and the matching oligonucleotides are assembled, thereby obtaining a first reaction product comprising at least one overhang. Further reaction products are then likewise obtained and are assembled in a predetermined workflow using an algorithm, thereby producing said target ds polynucleotide with an overhang, optionally followed by a finalization step to prepare blunt ends.

A method for determining the number of nucleic acid molecules (NAMs) in a group of NAMs, comprising i) obtaining an amplified and mutagenized group of NAMs that was produced by a. subjecting the group of NAMs to a chemical mutagenesis which mutates only select nucleic acid bases in the group of NAMs at a rate of 10% to 90% thus forming a group of mutagenized NAMs (mNAMs), and b. amplifying the group of mNAMs; ii) obtaining sequences of the mNAMs in the group of amplified mNAMs; and iii) counting the number of different sequences obtained in step (ii) to determine the number of unique mNAMs in the group of mNAMS,
thereby determining the number of NAMs in the group of NAMs.

A method for producing a dicarboxylic acid is provided. A dicarboxylic acid is produced by culturing a bacterium having a dicarboxylic acid-producing ability, which has been modified so that the expression of one or more of the yeeA gene, ynfM gene, yjjP gene, and yjjB gene is increased, in a medium, and collecting the dicarboxylic acid from the medium.

Provided herein are methods for the assembly of a polynucleic acid sequence that is at least partially carried out on a microfluidic device; methods for the preparation of a library of polynucleic acid sequences; microfluidic devices; methods for designing nucleic acid sequences; methods for planning the assembly of a polynucleic acid sequence from a plurality of nucleic acid sequences; systems comprising components for carrying out these methods; computer programs which, when run on a computer, implements these methods; and computer readable medium or carrier signals encoding such a computer program.

Aspects of the invention relate to methods, compositions for synthesizing oligonucleotides having a predefined sequence.

A method for increasing the cell surface expression and/or reducing mispairing of a TCR.

The present invention provides methods for generating a library of synthetic polynucleotides. The present invention also provides methods for generating proteins encoded by the library of synthetic polynucleotides. In addition, provided herein are methods for determining the soluble expression of said proteins. This invention is based, in part, on the discovery of a method for selecting optimal oligonucleotides in combination with performing a phosphorylation reaction, ligation reaction and PCR amplification in a single reaction vessel to produce synthetic polynucleotides in a multiplex manner.

Disclosed is a method of generating a set of sequence-verified nucleic acid elements for the combinatorial construction of genetic elements. The method includes: providing a plurality of nucleic acid parts; assembling nucleic acid parts to form a one or more nucleic acid elements, wherein the nucleic acid elements include at least two sequences selected from the plurality of parts; and determining the sequence of the nucleic acid elements. Further disclosed is pool of higher-order nucleic acid constructs or amplification products thereof, comprising one or more nucleic acid elements as well as kits including a the pool of sequence-verified nucleic acid elements of claims and/or a pool of higher-order nucleic acid constructs; and a plurality of primers for retrieving one or more sequence-verified nucleic acid elements and/or higher-order nucleic acid constructs.

Disclosed is a method of generating a set of sequence-verified nucleic acid elements for the combinatorial construction of genetic elements. The method includes: providing a plurality of nucleic acid parts; assembling nucleic acid parts to form a one or more nucleic acid elements, wherein the nucleic acid elements include at least two sequences selected from the plurality of parts; and determining the sequence of the nucleic acid elements. Further disclosed is pool of higher-order nucleic acid constructs or amplification products thereof, comprising one or more nucleic acid elements as well as kits including a the pool of sequence-verified nucleic acid elements of claims and/or a pool of higher-order nucleic acid constructs; and a plurality of primers for retrieving one or more sequence-verified nucleic acid elements and/or higher-order nucleic acid constructs.