Abstract: An adenovirus expression vector is provided. The adenovirus expression vector may include: a) one or more mutations that render the adenovirus replication incompetent and b) at least one nucleotide sequence encoding a protein or an RNA is provided. A method of synthesizing an adenovirus vector is also provided. The synthesis may include: a) producing a plurality of overlapping adenovirus sub-fragments, each sub-fragment comprising a portion of the full genome of the adenovirus; b) circularizing the sub-fragments to form plasmid structures; and c) assembling the circularized sub-fragments into a linear structure, wherein the vector comprises a combination of two or more sub-fragments. A mammalian cell line configured to replicate adenoviral vectors, wherein the cell line comprises nucleotide sequences expressing E1A and E1B gene products but is devoid of other adenovirus sequences is also provided.

Abstract: The present invention provides methods and compositions for the generation and identification of expression constructs that can be used to express sufficient levels of a polypeptide of interest. The compositions include a population of expression vectors, wherein members of the population have a type IIS restriction enzyme recognition site adjacent to a regulatory sequence, and wherein the regulatory element is distinct in at least two members of the population of expression vectors. In various embodiments, the expression vectors further comprise a polynucleotide sequence encoding a polypeptide of interest, wherein the polynucleotide encoding the polypeptide, the polynucleotide of the regulatory sequence, or both, are distinct in at least two members of the population. The compositions are useful for identifying a combination of coding sequences and/or regulatory elements useful for the heterologous expression of the polypeptide of interest.