Methods and means for producing efficient silencing construct using recombinational cloning
First Claim
Patent Images
1. A vector comprising the following operably linked DNA fragments:
- an origin of replication allowing replication in a recipient cell (1), a selectable marker region (2) capable of being expressed in said recipient cell; and
a chimeric DNA construct comprising in sequence;
a promoter or promoter region (3) capable of being recognized by RNA polymerases of a eukaryotic cell;
a first recombination site (4), a second recombination site (5), a third recombination site (6) and a fourth recombination site (7);
a 3′
transcription terminating and polyadenylation region (8) functional in said eukaryotic cell;
wherein said first recombination site (4) and said fourth recombination site (7) are capable of reacting with a same recombination site; and
said second recombination site (5) and said third recombination site (6), are capable of reacting with a same recombination site; and
wherein said first recombination site (4) and said second recombination site (5) do not recombine with each other or with a same recombination site;
or said third recombination site (6) and said fourth recombination site (7) do not recombine with each other or with a same recombination site.
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Abstract
Methods and vectors and kits are provided for producing chimeric nucleic acid constructs capable of producing dsRNA for silencing target nucleic acid sequences of interest using recombinational cloning.
56 Citations
28 Claims
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1. A vector comprising the following operably linked DNA fragments:
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an origin of replication allowing replication in a recipient cell (1), a selectable marker region (2) capable of being expressed in said recipient cell; and
a chimeric DNA construct comprising in sequence;
a promoter or promoter region (3) capable of being recognized by RNA polymerases of a eukaryotic cell;
a first recombination site (4), a second recombination site (5), a third recombination site (6) and a fourth recombination site (7);
a 3′
transcription terminating and polyadenylation region (8) functional in said eukaryotic cell;
whereinsaid first recombination site (4) and said fourth recombination site (7) are capable of reacting with a same recombination site; and
said second recombination site (5) and said third recombination site (6), are capable of reacting with a same recombination site; and
whereinsaid first recombination site (4) and said second recombination site (5) do not recombine with each other or with a same recombination site;
orsaid third recombination site (6) and said fourth recombination site (7) do not recombine with each other or with a same recombination site. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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13. A vector comprising the sequence of SEQ ID No 13.
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14. A vector comprising the sequence of SEQ ID No 23.
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15. A vector comprising the sequence of SEQ ID No 24.
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16. A vector comprising the sequence of SEQ ID No 25.
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17. A vector comprising the sequence of SEQ ID No 26.
Specification