The invention provides, inter alia, a nucleic acid (e.g. expression vector) that comprises at least a first coding sequence and a second coding sequence. Each conding sequence is under the control of an inducible promoter of defined strength. Different promoters can have different strengths. Each promoter is responsive to the same inducer. The invention also provides: methods of expressing coding regions, methods of making a product of a multi-enzyme pathway, and methods of optimizing the yield of a product of a multi-enzyme metabolic pathway using the nucleic acids provided by the invention. Also disclosed is a method of non-enzymatic gene cloning useful for practicing the invention.

The invention provides a bacterial expression vector (100) comprising of a secretory signal sequence in tandem with DNA sequence encoding recombinant protein (103), wherein, the secretory signal sequence is a combination comprising of: a) at least one DNA sequence encoding a signal sequence (101) of gene selected from the group consisting of pelB, ompA, yebF, and ompF, and b) at least one DNA sequence encoding a carrier peptide (102) selected from the group consisting of Seq. ID 5 and 6 encoding truncated yebF.

The invention provides, inter alia, a nucleic acid (e.g. expression vector) that comprises at least a first coding sequence and a second coding sequence. Each conding sequence is under the control of an inducible promoter of defined strength. Different promoters can have different strengths. Each promoter is responsive to the same inducer. The invention also provides: methods of expressing coding regions, methods of making a product of a multi-enzyme pathway, and methods of optimizing the yield of a product of a multi-enzyme metabolic pathway using the nucleic acids provided by the invention. Also disclosed is a method of non-enzymatic gene cloning useful for practicing the invention.

The present invention relates to a Plasmid Addiction System for the stabilization of expression plasmids encoding proteins of interest. The invention uses a succinate cycle optimization to ensure the expression of plasmid(s) of interest. By ensuring that plasmids of interest contain genes necessary in the succinate cycle, the system ensures that the passage of the plasmid to daughters and therefore improves the efficiency of production and expression of genes and/or products of interest.

Provided herein, in some aspects, are methods and compositions for cell-free production of ribonucleic acid.

The present disclosure provides methods and compositions that include a polynucleotide that includes nucleic acids that encode an anti-idiotype polypeptide, as well as polypeptides that are encoded by the same, and cells that include and express the polypeptide. Disclosed methods include methods that utilize the anti-idiotype polypeptides as safety switches when they are used in combination with antibodies, include approved biologic antibodies, that include the recognized idiotype. Certain embodiments include anti-idiotype polypeptides and nucleotides encoding the same, that include an internal domain. This internal domain in some embodiments has functional domains that can induce proliferation or cell death upon binding of the anti-idiotype polypeptide by its target antibody.

The invention provides compositions and methods for manufacturing pluripotent cells. In particular, the invention provides improved culture platforms for manufacturing pluripotent cells with ground state pluripotency.

The present invention relates to a Plasmid Addiction System for the stabilization of expression plasmids encoding proteins of interest. The invention uses a succinate cycle optimization to ensure the expression of plasmid(s) of interest. By ensuring that plasmids of interest contain genes necessary in the succinate cycle, the system ensures that the passage of the plasmid to daughters and therefore improves the efficiency of production and expression of genes and/or products of interest.

The invention provides compositions and methods for manufacturing pluripotent cells. In particular, the invention provides improved culture platforms for manufacturing pluripotent cells with ground state pluripotency.

The present invention relates to a Plasmid Addiction System for the stabilization of expression plasmids encoding proteins of interest. The invention uses a succinate cycle optimization to ensure the expression of plasmid(s) of interest. By ensuring that plasmids of interest contain genes necessary in the succinate cycle, the system ensures that the passage of the plasmid to daughters and therefore improves the efficiency of production and expression of genes and/or products of interest.