Abstract: Methods for the creation of one or more user-defined mutations that can be located anywhere in a target sequence, such as in a gene are disclosed. These methods can be used on a single-stranded or double-stranded template and, for a single-stranded template, generally include: (a) conducting a first amplification reaction in the presence of a thermostable DNA polymerase and a thermostable DNA ligase to synthesize a mutagenized strand of DNA comprising at least one mutagenic oligonucleotide relative to a complementary single-stranded template DNA comprising a target nucleic acid molecule in a circular DNA vector; (b) conducting a second amplification reaction in the presence of a thermostable DNA polymerase and a thermostable DNA ligase to synthesize a complementary mutant strand of DNA; and (c) degrading the template DNA and non-covalently closed circular nucleic acid molecules to obtain a mutation-containing double-stranded DNA product.

Abstract: The presently disclosed subject matter provides methods for the creation of one or more user-defined mutations that can be located anywhere in a target sequence, such as in a gene. These mutations can comprise single mutations, multiple mutations, or a comprehensive codon mutagenesis library, in which all possible single codon substitutions in a gene are created. These methods can be used on a single-stranded or double-stranded template.

Abstract: The presently disclosed subject matter relates to modified Kunkel mutagenesis methods that use a thermostable DNA polymerase and a thermostable DNA ligase.

Abstract: The presently disclosed subject matter relates to modified Kunkel mutagenesis methods that use a thermostable DNA polymerase and a thermostable DNA ligase.

Abstract: The invention provides molecular switches which couple external signals to functionality, and combinatorial methods of making and using the same involving circular permutation of nucleic acid and amino acid sequences. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches, expression vectors and host cells for expressing the switches are also provided.

Abstract: The presently disclosed subject matter provides methods for the creation of one or more user-defined mutations that can be located anywhere in a target sequence, such as in a gene. These mutations can comprise single mutations, multiple mutations, or a comprehensive codon mutagenesis library, in which all possible single codon substitutions in a gene are created. These methods can be used on a single-stranded or double-stranded template.

Abstract: The invention provides molecular switches which couple external signals to functionality, and combinatorial methods of making and using the same involving circular permutation of nucleic acid and amino acid sequences. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches, expression vectors and host cells for expressing the switches are also provided.

Abstract: The present invention features a novel protein engineering strategy by combining the domains of two independent proteins into a molecular switch. The invention features polypeptides comprising a prodrug activating enzyme and a protein that binds a cancer specific marker, polynucleotides encoding the polypeptides, and molecular switches for converting a prodrug into a toxin, comprising the polypeptides. The invention also features methods for converting a prodrug into a toxin, methods for treating cancer, and methods for making the molecular switches, as well as kits.

Abstract: The invention provides molecular switches which couple external signals to functionality, and combinatorial methods of making and using the same involving circular permutation of nucleic acid and amino acid sequences. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches, expression vectors and host cells for expressing the switches are also provided.

Abstract: The presently disclosed subject matter provides methods for the creation of one or more user-defined mutations that can be located anywhere in a target sequence, such as in a gene. These mutations can comprise single mutations, multiple mutations, or a comprehensive codon mutagenesis library, in which all possible single codon substitutions in a gene are created. These methods can be used on a single-stranded or double-stranded template.

Abstract: The present invention features a novel protein engineering strategy by combining the domains of two independent proteins into a molecular switch. The invention features polypeptides comprising a prodrug activating enzyme and a protein that binds a cancer specific marker, polynucleotides encoding the polypeptides, and molecular switches for converting a prodrug into a toxin, comprising the polypeptides. The invention also features methods for converting a prodrug into a toxin, methods for treating cancer, and methods for making the molecular switches, as well as kits.