Nucleic acid molecule comprising a coding sequence and a region of increased folding energy upstream of a stop codon are provided. Expression vectors and cells comprising the nucleic acid molecule are also provided. Methods for optimizing a coding sequence comprising increasing folding energy in a region upstream of the stop codon are also provided.

According to one embodiment, a first gene encodes a reporter protein. The first gene is disposed at the downstream of the gene promoter. A second gene is disposed at the downstream of the gene promoter and encodes a replication origin-binding protein. An internal ribosome entry site is disposed between the first gene and the second gene. The transcription termination signal sequence encodes a signal for terminating the transcription of the first gene and the second gene. A replication origin sequence is recognized by the replication origin-binding protein.

According to one embodiment, a first gene encodes a reporter protein. The first gene is disposed at the downstream of the gene promoter. A second gene is disposed at the downstream of the gene promoter and encodes a replication origin-binding protein. An internal ribosome entry site is disposed between the first gene and the second gene. The transcription termination signal sequence encodes a signal for terminating the transcription of the first gene and the second gene. A replication origin sequence is recognized by the replication origin-binding protein.

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, 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.

Gene-therapeutic DNA vectors based on the VTvaf17 gene-therapeutic DNA vector have been created for the treatment of diseases characterized by impaired mucociliary transport and mucolytic function and the development of mucostasis, including cystic fibrosis. The gene therapy DNA vector contains the coding region of the CFTR therapeutic gene, or the NOS1 therapeutic gene, or the AQ1 therapeutic gene, or the AQ3 therapeutic gene, or the AQ5 therapeutic gene. Methods for their preparation or use are proposed, as well as strains for the production of a gene therapy vector.

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, 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 disclosure is directed to an expression vector or a combination of at least two expression vectors for producing a polypeptide of interest, the vector or vectors comprising a polynucleotide encoding a mutated folate receptor as a selectable marker. The disclosure also relates to host cells, selection methods and methods for producing polypeptides with high yield.

The disclosure is directed to an expression vector or a combination of at least two expression vectors for producing a polypeptide of interest, the vector or vectors comprising a polynucleotide encoding a mutated folate receptor as a selectable marker. The disclosure also relates to host cells, selection methods and methods for producing polypeptides with high yield.