TAL-effector assembly platform, customized services, kits and assays
The invention generally relates to compositions and methods for designing and producing functional DNA binding effector molecules and associated customized services, tool kits and functional assays. In some aspects, the invention provides methods and tools for efficient assembly of customized TAL effector molecules. Furthermore, the invention relates to uses of TAL effector molecules and functional evaluation of such TAL by, for example, customized assays.
1. A nucleic acid molecule encoding a non-naturally occurring DNA binding protein, the non-naturally occurring DNA binding protein comprising:
(a) an amine terminal region of between 25 and 500 amino acids,
(b) a carboxyl terminal region of between 25 and 500 amino acids, and
(c) a central region containing five or more amino acid segments which confer upon the non-naturally occurring protein sequence specific nucleic acid binding activity,
wherein the individual amino acid segments comprise the amino acid sequence Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Xaa1 Xaa2 Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly, wherein Xaa1 and Xaa2 are His Asp, Asn Ile, Asn Lys, or Asn Gly (SEQ ID NO: 234),
wherein the N-terminus of the non-naturally occurring DNA binding protein comprises a V5 epitope,
wherein the non-naturally occurring DNA binding protein comprises a nuclear localization sequence comprising the amino acid sequence of SEQ ID NO: 107, and
wherein at least two of the individual amino acid segments are encoded by nucleic acids wherein different codons encode the same amino acids.
2. The nucleic acid molecule of claim 1 , wherein the V5 epitope comprises the amino acid sequence of SEQ ID NO: 88.
3. The nucleic acid molecule of claim 2 , wherein at least two of the individual amino acid segments encoded by nucleic wherein different codons encode the same amino acids comprise primer binding sites not present elsewhere in the nucleic acid molecule.
4. The nucleic acid molecule of claim 1 , wherein one of the individual amino acid segments is encoded by nucleic acid comprising the nucleotide sequence of SEQ ID NO: 72.
5. The nucleic acid molecule of claim 1 , wherein the non-naturally occurring DNA binding protein further comprises an effector domain.
6. The nucleic acid molecule of claim 5 , wherein the effector domain is a nuclease domain.
7. A vector comprising the nucleic acid molecule of claim 1 .
8. The vector of claim 7 , wherein the nucleic acid molecule is operably connected to a Cytomegalovirus (CMV) promoter.
9. A host cell comprising the nucleic acid molecule of claim 1 .
10. A protein encoded by the nucleic acid molecule of claim 1 .
11. A method for genetic manipulation within a cell, the method comprising introducing the nucleic acid molecule of claim 1 into the cell under conditions that allow for intracellular production of the non-naturally occurring DNA binding protein.