Viral open reading frames, uses thereof, and methods of detecting the same

A vector comprising:

• a synthetic library expression construct comprising:
• a short synthetic polynucleotide;
• a pair of constant primers flanking the short synthetic polynucleotide, the pair of constant primers comprising a forward constant primer and a reverse constant primer, wherein the forward constant primer and the reverse constant primer are each independently coupled to a 5′ end or a 3′ end of the short synthetic polynucleotide;
• a stop codon polynucleotide comprising one or more stop codons, wherein the stop codon polynucleotide is coupled to a constant primer coupled to the 3′ end of the short synthetic polynucleotide such that the constant primer coupled to the 3′ end of the short synthetic polynucleotide is between the stop codon polynucleotide and the short synthetic polynucleotide;
• a poly A signal, wherein the poly A signal is operably coupled to the short synthetic polynucleotide, the pair of constant primers, and the stop codon polynucleotide;
• and
• a promoter, wherein the promoter is operably coupled to the short synthetic polynucleotide, the pair of constant primers, and the stop codon polynucleotide, and the poly A signal.

A method of determining translational regulation comprising:

• determining a first set of translated sequences by a method comprising:
• expressing a pan-genomic library in a first plurality of cells, wherein the pan-genomic library comprises a plurality of synthetic library expression constructs, each comprising a short synthetic polynucleotide and a pair of constant primers flanking the short synthetic polynucleotide, wherein the short synthetic polynucleotide has a sequence corresponding to a region of a genome;
• obtaining a sample of ribosomes comprising ribosome-protected mRNA fragments (RPFs) from the first plurality of cells;
• recovering ribosome footprints consisting essentially of the RPFs from the sample of ribosomes;
• and
• determining the sequence of the RPFs thereby identifying translated sequences to form the first set of translated sequences;
• determining a second set of translated sequences by a method comprising:
• expressing the pan-genomic library in a second plurality of cells;
• applying a stress to the second plurality of cells;
• obtaining a sample of ribosomes comprising ribosome-protected mRNA fragments (RPFs) from the second plurality of cells;
• recovering ribosome footprints consisting essentially of the RPFs from the sample of ribosomes;
• and
• determining the sequence of the RPFs thereby identifying translated sequences to form the second set of translated sequences,
• whereby similarities and differences in the sequences of the first and the second set of translated sequences indicates sequences that are translationally regulated or regulate translation.

A vector comprising:

• an optically active reporter construct comprising:
• a test polynucleotide;
• a first optically active protein encoding polynucleotide, wherein the first optically active protein encoding polynucleotide is operatively coupled in-frame with the test polynucleotide;
• a second optically active protein encoding polynucleotide, wherein the second optically active protein is a different optically active protein that the first optically active protein;
• a promoter, wherein the test polynucleotide, the first optically active protein encoding polynucleotide, and the second optically active protein encoding polynucleotide are operatively coupled to the promoter;
• and
• an internal ribosomal entry site (IRES), wherein the IRES is between the first optically active protein encoding polynucleotide and the second optically active protein encoding polynucleotide.