The present invention relates to a nucleic acid sequence comprising a nucleotide of interest and a tryptophan RNA-binding attenuation protein (TRAP) binding site, and optionally a Kozak sequence, wherein said TRAP binding site overlaps the Kozak sequence and/or the ATG start codon of the nucleotide of interest. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest and a Kozak sequence, wherein said Kozak sequence comprises a portion of a tryptophan RNA-binding attenuation protein (TRAP) binding site. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest and TRAP binding site wherein the TRAP binding site comprises a portion of the start codon ATG of said nucleotide of interest or wherein the ATG start codon comprises a portion of the TRAP binding site. The present invention further relates to a nucleic acid sequence comprising a nucleotide of interest, a binding site for tryptophan RNA-binding attenuation protein (TRAP), a multiple cloning site and a Kozak sequence, wherein said multiple cloning site is overlapping with or located downstream to the 3′ KAGN2-3 repeat of the TRAP binding site and upstream of the Kozak sequence.

Recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

The present invention discloses methods and compositions for modulating the quality of an immune response to a target antigen in a mammal, which response results from the expression of a polynucleotide that encodes at least a portion of the target antigen, wherein the quality is modulated by replacing at least one codon of the polynucleotide with a synonymous codon that has a higher or lower preference of usage by the mammal to confer the immune response than the codon it replaces.

Described herein are methods and compositions for producing a gene of interest (GOD, which, in certain embodiments, can reduce the metabolic burden on cells and reduce decoupling of GOI production from marker production, as compared to prior art methods. The methods relate to positive selection and negative selection approaches to establishing high GOI-producing cell lines, e.g., CHO lines. In certain embodiments, the methods comprise transfecting a cell with (a) an oligonucleotide comprising a GOI and a non-coding RNA, and (b) an oligonucleotide encoding a selection protein; wherein the non-coding RNA promotes or inhibits production of the selection protein. The cell producing the can be identified and/or selected as a result of or by detecting the absence or the presence of the selection protein.

Described herein are methods and compositions for producing a gene of interest (GOI) which, in certain embodiments, can reduce the metabolic burden on cells and reduce decoupling of GOI production from marker production, as compared to prior art methods. The methods relate to positive selection and negative selection approaches to establishing high GOI-producing cell lines, e.g., CHO lines. In certain embodiments, the methods comprise transfecting a cell with (a) an oligonucleotide comprising a GOI and a non-coding RNA, and (b) an oligonucleotide encoding a selection protein; wherein the non-coding RNA promotes or inhibits production of the selection protein. The cell producing the GOI can be identified and/or selected as a result of or by detecting the absence or the presence of the selection protein.

Engineered synthetic RNA-based genetic circuits are provided that are regulated exclusively at the post-transcriptional level.

Aspects of the disclosure relate to compositions and methods for expressing one or more Ganglioside GM3 synthase (GM3S) isoforms in a cell or subject. In some aspects, the disclosure relates to methods for treating GM3 synthase deficiency in a subject in need thereof.

Engineered synthetic RNA-based genetic circuits are provided that are regulated exclusively at the post-transcriptional level.

The invention in some aspects relates to isolated nucleic acids, compositions, and kits useful for identifying adeno-associated viruses in cells. In some aspects, the invention provides kits and methods for producing somatic transgenic animal models using recombinant AAV (rAAV) to an animal having at least one transgene that expresses a small interfering nucleic acid or at least one binding site for a miRNA.

The invention provides adeno-associated virus (AAV) replication (Rep) sequences. In one embodiment, the invention provides nucleotide sequences encoding a chimeric protein, wherein the encoded chimeric protein contains a wild type AAV Rep inhibitory amino acid sequence, and wherein the nucleotide sequences contain a scrambled and/or deoptimized polynucleotide sequence encoding the wild type AAV Rep inhibitory amino acid sequence. The invention provides vectors, cells, and viruses containing the invention's sequences. Also provided are methods for detecting portions of the AAV Rep inhibitory amino acid sequence, which reduce replication and/or infection and/or productive infection by viruses. The invention's compositions and methods are useful for site-specific integration and/or expression of heterologous sequences by recombinant adeno-associated virus (rAAV) vectors and by rAAV virus particles, such as hybrid viruses (e.g., Ad-AAV) comprising such vectors. The invention's compositions and methods find application in, for example, gene therapy and/or vaccines.