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US 9,040,677 B2Granted
Transcription activator-like effector assembly
AssigneeSIDANSAI Biotechnology CO., LTD
InventorsJinlong Zhao, Zhao Wu
Life of this patent
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Abstract
Described herein are techniques for assembling a polynucleotide encoding a transcription activator-like effector nucleases (TALEN). The techniques ligate and digest necessary modules for a TALEN assembly in one reactor or system. Methods and Kits for generating a TALEN are also described.
Claims16
A method for assembling a polynucleotide encoding a transcription activator-like effector (TALE) polypeptide, the method comprising:
- generating multiple Deoxyribonucleic acid (DNA) segments, an individual DNA segment of the multiple DNA segments corresponding to a repeat sequence of the TALE polypeptide, the repeat sequence identifying a single nucleotide of a particular target polynucleotide or two contiguous nucleotides of the particular target polynucleotide;
- mixing the multiple DNA segments with restriction enzymes, DNA ligases, and a TALE backbone vector to generate the polynucleotide encoding the TALE polypeptide, a number of multiple DNA segments being greater than 14;
- and
- purifying the polynucleotide using a plasmid-safe Deoxyribonuclease (DNase).
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A kit for generating polynucleotides encoding a transcription activator-like effector (TALE) polypeptide, the kit comprising:
- multiple sets of dimer DNA segments, an individual set of dimer DNA segments including multiple dimer DNA segments, an individual dimer DNA segment encoding two recognition domains of a designed TALE that identify two contiguous nucleotides of a particular target polynucleotide;
- multiple sets of monomer DNA segments, an individual set of monomer DNA segments including multiple monomer DNA modules, an individual monomer DNA module encoding a recognition domain of the designed TALE that identifies a single nucleotide of the particular target polynucleotide, wherein the individual dimer DNA segments and the individual monomer DNA segments including a first overhang and a second overhang that are generated using a type II restriction endonuclease;
- restriction enzymes;
- DNA ligases;
- and
- TALE backbone vectors.
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Dependencies are inferred from the claim text.