The present invention encompasses compositions and methods to allow one to deliver and express multiple genes from a biosynthetic pathway in a recipient cell via a synthetic chromosome.

The present invention encompasses compositions and methods to allow one to deliver and express multiple genes from a biosynthetic pathway in a recipient cell via a synthetic chromosome.

Fungal artificial chromosome (FAC) vectors are disclosed. A vector can be replicated in a bacterial or a fungal host, and can comprise an insert of heterologous DNA up to about 500 kilobases. A vector can be used for cloning and expressing a secondary metabolite (SM) gene cluster. An insert sequence can be modified by homologous recombination. A vector can be a plasmid comprising bacterial and fungal origins of replication, as well as bacterial and fungal selection marker genes. Also disclosed are vectors that can be integrated into a fungal genome, and dual function vectors which can be replicated in a bacterial or a fungal host and can also be integrated into a fungal genome. Also disclosed are methods of generating plasmid libraries including vectors comprising intact SM gene clusters.

Fungal artificial chromosome (FAC) vectors are disclosed. A vector can be replicated in a bacterial or a fungal host, and can comprise an insert of heterologous DNA up to about 500 kilobases. A vector can be used for cloning and expressing a secondary metabolite (SM) gene cluster. An insert sequence can be modified by homologous recombination. A vector can be a plasmid comprising bacterial and fungal origins of replication, as well as bacterial and fungal selection marker genes. Also disclosed are vectors that can be integrated into a fungal genome, and dual function vectors which can be replicated in a bacterial or a fungal host and can also be integrated into a fungal genome. Also disclosed are methods of generating plasmid libraries including vectors comprising intact SM gene clusters.

Fungal artificial chromosome (FAC) vectors are disclosed. A vector can be replicated in a bacterial or a fungal host, and can comprise an insert of heterologous DNA up to about 500 kilobases. A vector can be used for cloning and expressing a secondary metabolite (SM) gene cluster. An insert sequence can be modified by homologous recombination. A vector can be a plasmid comprising bacterial and fungal origins of replication, as well as bacterial and fungal selection marker genes. Also disclosed are vectors that can be integrated into a fungal genome, and dual function vectors which can be replicated in a bacterial or a fungal host and can also be integrated into a fungal genome. Also disclosed are methods of generating plasmid libraries including vectors comprising intact SM gene clusters.

Aspects of the present invention relate to synthetic chromosomes that may be incorporated into leukocytes, wherein the synthetic chromosomes comprise nucleic acid sequences encoding multiple Chimeric Antigen Receptors (CARs). Such manipulated leukocytes can be used in medicine, notably in the treatment of a cancer such as in the treatment of cancer having solid tumors. The leukocytes may be lymphocytes, including tumor-infiltrating lymphocytes. T cells. NK cells or B cells. In a preferred aspect, the leucocytes are syngeneic and T cells.

The disclosure provides cleavable closed-ended DNA (ceDNA). In some embodiments, the cleavable ceDNA may be used as donor or repair template for editing of a target sequence in the genome.