The present disclosure relates to materials and methods for spatially analyzing nucleic acids that have been fragmented with a transposase enzyme, alone or in combination with other types of analytes.

The present invention provides hyperactive piggyBac transposons, in particular hyperactive piggyBac transposons from Trichoplusia ni (cabbage looper moth) that transpose at a higher frequency than wildtype. The invention also features integration defective piggyBac transposons. The piggyBac transposons and transposases can be used in gene transfer systems for stably introducing nucleic acids into the DNA of a cell. The gene transfer system can be used in methods, for example, but not limited to, gene therapy, insertional mutagenesis, or gene discovery.

Provided herein are methods and compositions for synthesizing 5′Capped RNAs wherein the initiating capped oligonucleotide primers have the general form .sup.m7Gppp[N.sub.2′Ome].sub.n[N].sub.m wherein .sup.m7G is N7-methylated guanosine or any guanosine analog, N is any natural, modified or unnatural nucleoside, “n” can be any integer from 0 to 4 and “m” can be an integer from 1 to 9.

The present invention provides polynucleotide vectors for high expression of heterologous genes. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, gene expression, bioprocessing, gene therapy, insertional mutagenesis, or gene discovery.

Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Such methods, systems, and compositions for seamless nucleic acid assembly include those for in vitro recombination cloning, single-stranded hierarchal DNA assembly, or overlap extension PCR without primer removal.

This disclosure provides various TcBuster transposases and transposons, systems, and methods of use.

Provided herein, among other things, is a conjugate comprising a polymerase and a nucleoside triphosphate, where the polymerase and the nucleoside triphosphate are covalently linked via a linker that comprises a cleavable linkage. A set of such conjugates, where the conjugates correspond to G, A, T (or U) and C is also provided. Methods for synthesizing a nucleic acid of a defined sequence are also provided. The conjugates can also be used for sequencing applications.

Provided herein are methods and compositions for synthesizing 5′Capped RNAs wherein the initiating capped oligonucleotide primers have the general form .sup.m7Gppp[N.sub.2′Ome].sub.n[N].sub.m wherein .sup.m7G is N7-methylated guanosine or any guanosine analog, N is any natural, modified or unnatural nucleoside, “n” can be any integer from 0 to 4 and “m” can be an integer from 1 to 9.

Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Such methods, systems, and compositions for seamless nucleic acid assembly include those for in vitro recombination cloning, single-stranded hierarchal DNA assembly, or overlap extension PCR without primer removal.

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using renewable initiators coupled to a solid support. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template.