The present invention relates to a method for constructing DNA molecules, comprising: contacting a plurality of double-stranded DNA fragments with a cell-free extract of a yeast strain in a single in vitro reaction to combine the plurality of DNA fragments into the DNA molecules, wherein each of the DNA fragments has a 5′ end and a 3′ end, and wherein the DNA fragments combine with each other when the 5′ end of one fragment has at least 15 bp that are homologous with the 3′ end of another fragment.

The present invention features modified polynucleotide sequences that mimic host cell DNA and methods of using such sequences for the genetic engineering of bacteria that are otherwise genetically intractable.

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization from a single cell. Such polynucleotide processing may be useful for a variety of applications. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins) and chromatin (e.g., accessible chromatin).

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization from one or more cells. Such polynucleotide processing may be useful for a variety of applications, including generation of labeled macromolecules, including major mistocompatability complex (MHC) molecules, dextramers, etc. Labeled macromolecules may be generated using an in vitro transcription reaction. Labeled macromolecules may be generated in one or more partitions.

In certain embodiments, this disclosure relates to conjugates comprising a polypeptide of GM-CSF and a polypeptide IL-4. Typically, the GM-CSF and IL-4 are connected by a linker, e.g., polypeptide. In certain embodiments, the disclosure relates to isolated nucleic acids encoding these polypeptide conjugates, vectors comprising nucleic acid encoding polypeptide conjugates, and protein expression systems comprising these vectors such as infectious viral particles and host cells comprising such nucleic acids.

Methods for generating synthetic genomes, for example synthetic genomes having desired properties or viable genomes of reduced size, are disclosed. Also disclosed are synthetic genomes produced by the methods disclosed herein and synthetic cells containing the synthetic genomes disclosed herein.

The invention provides a process for synthesizing genes and other long double stranded polynucleotides by assembling very short oligonucleotides into partly double stranded polynucleotides, and then connecting these partly double stranded polynucleotide subassemblies with linkers comprised of very short oligonucleotides. In one embodiment, the correct order of the polynucleotide subassemblies is coded in overhangs present at each end of the partly double stranded polynucleotide subassemblies. Linkers having a sequence complimentary to the combined overhangs connect adjacent subassemblies, which are then ligated together. In one preferred embodiment the oligos are six bases long, for which there are only 4096 different possible sequence permutations. A complete library of oligos of this size and scale can be cost-effectively synthesized and quality controlled, avoiding the typical errors and yield issues associated with phosphoramidite synthesis of longer oligos. Furthermore, the limited oligo library size supports development of a laboratory-scale gene synthesis machine.

The present invention relates to a rapid, high-fidelity process to support synthesis of genes for in vitro transcription of modified messenger RNA. In this process, sequence errors resulting from amplification with oligonucleotides comprising inherent errors are significantly reduced.

Disclosed herein are methods for the generation of highly accurate nucleic acid libraries encoding for predetermined variants of a nucleic acid sequence. The nucleic acid sequence may encode for all or part of a reference domain of a CAR. The degree of variation may be complete, resulting in a saturated variant library, or less than complete, resulting in a non-saturating library of variants. The variant nucleic acid libraries described herein may be designed for further processing by transcription or translation. The variant nucleic acid libraries described herein may be designed to generate variant RNA, DNA and/or protein populations. Further provided herein are method for identifying variant species with increased or decreased activities, with applications in regulating biological functions and the design of therapeutics for treatment or reduction of a disease, such as cancer.

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing and analyte characterization from a single cell. Such polynucleotide processing may be useful for a variety of applications. The compositions, methods, systems, and devices disclosed herein generally describe barcoded oligonucleotides, which can be bound to a bead, such as a gel bead, useful for characterizing one or more analytes including, for example, protein (e.g., cell surface or intracellular proteins) and chromatin (e.g., accessible chromatin).