US 11,597,937 B2Granted

System and method of modular cloning

AssigneeIcon Genetics GmbH

InventorsErnst Weber, Stefan Werner, Carola Engler, Ramona Grutzner, Sylvestre Marillonnet

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Abstract

System for producing a nucleic acid construct of interest, said system comprising: a set of n entry DNAs numbered 1 to n, n being an integer of at least 2, each of said n entry DNAs comprising in this order: (i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof; (ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and (iii) a cleavage site of a further type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site; the cleavage sites of the type IIs restriction endonuclease recognition sites of item (iii) of entry DNAs 1 to n−1 are complementary to the cleavage sites of the type IIs restriction endonuclease recognition sites of item (i) of entry DNAs 2 to n, respectively; the cleavage site of the type IIs restriction endonuclease recognition site of item (iii) of entry DNA n is complementary to the cleavage site of the type IIs restriction endonuclease recognition site of item (i) of entry DNA 1 for allowing annealing of complementary single-stranded overhangs formed by restriction at recognition site (i) of entry DNA 1 and at recognition site (iii) of entry DNA n; said system further comprising a destination vector comprising in this order: (I) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof; (II) a vector backbone preferably comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III); (III) a further cleavage site of a type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site, and (IV) optionally, an insert between the recognition sites of item (III) and item (I); said cleavage sites of items (I) and (III) being different and non-complementary, said recognition sites of items (I) and (III) being preferably recognitions sites of the same endonuclease.

Claims12

Dependent claims

System for producing a nucleic acid construct of interest, said system comprising:

a set of n entry DNAs numbered 1 to n, n being an integer of at least 3,
each of said n entry DNAs comprising in this order:
(i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;
(ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and
(iii) a cleavage site of a further type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site;
the cleavage sites of the type IIs restriction endonuclease recognition site(s) of item (iii) of entry DNA(s) 1 to n−1 is/are complementary to the cleavage site(s) of the type IIs restriction endonuclease recognition site(s) of item (i) of entry DNA(s) 2 to n, respectively;
the cleavage site of the type IIs restriction endonuclease recognition site of item (iii) of entry DNA n is complementary to the cleavage site of the type IIs restriction endonuclease recognition site of item (i) of entry DNA 1;
said system further comprising a destination vector comprising in this order:
(I) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;
(II) a vector backbone comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III);
(III) a further cleavage site of a type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site, and
(IV) optionally, an insert between the recognition sites of item (III) and item (I);
said system further comprising a nucleic acid linker comprising in the following order:
(a) a type IIs restriction endonuclease recognition site;
(b) a cleavage site of said recognition site of item (a);
(c) a cleavage site of a further type IIs restriction endonuclease recognition site of the following item (d);
(d) a type IIs restriction endonuclease recognition site defining the cleavage site of item (c) and being a recognition site of a type IIs restriction endonuclease different from that of item (a);
(e) a type IIs restriction endonuclease recognition site;
(f) a cleavage site of said recognition site of item (e);
(g) a cleavage site of a further type IIs restriction endonuclease recognition site of the following item (h);
(h) a type IIs restriction endonuclease recognition site defining the cleavage site of item (g);
said linker being capable of linking a cleavage site of item (iii) of one of a entry DNA numbered 1 to n to a cleavage site of item (III) of said destination vector.

19 more elements

System for producing a nucleic acid construct of interest, said system comprising:

a set of n entry DNAs numbered 1 to n, n being an integer of at least 3,
each of said n entry DNAs comprising in this order:
(i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;
(ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and
(iii) a cleavage site of a further type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site;
the cleavage sites of the type IIs restriction endonuclease recognition sites of item (iii) of entry DNAs 1 to n−1 are complementary to the cleavage sites of the type IIs restriction endonuclease recognition sites of item (i) of entry DNAs 2 to n, respectively;
all cleavages sites of item (i) are unique among said n entry DNAs, and all cleavage sites of item (iii) are unique among said n entry DNAs;
said system further comprising a destination vector comprising in this order:
(I) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;
(II) a vector backbone comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III);
(III) a further cleavage site of a type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site, and
(IV) optionally, a linker between the recognition sites of item (III) and item (I);
said system further comprising a nucleic acid linker comprising in the following order:
(a) a type IIs restriction endonuclease recognition site;
(b) a cleavage site of said recognition site of item (a);
(c) a cleavage site of a further type IIs restriction endonuclease recognition site of the following item (d);
(d) a type IIs restriction endonuclease recognition site defining the cleavage site of item (c) and being a recognition site of a type IIs restriction endonuclease different from that of item (a);
(e) a type IIs restriction endonuclease recognition site;
(f) a cleavage site of said recognition site of item (e);
(g) a cleavage site of a further type IIs restriction endonuclease recognition site of the following item (h);
(h) a type IIs restriction endonuclease recognition site defining the cleavage site of item (g);
said linker being capable of linking a cleavage site of item (iii) of one of a entry DNA numbered 1 to n to a cleavage site of item (III) of said destination vector.

19 more elements

Dependencies are inferred from the claim text.

Facts

Application: 15/634,803Filed 27 Jun 2017 · division
Priority: EP 1000609011 Jun 2010
Published: US 2017/0369889 A128 Dec 2017
Granted: 7 Mar 2023
Term ends: Not estimatedThe source records don’t support an estimate.

Classification5 codes

C12N 15/66
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression: general methods for inserting a…
C12N 15/1093
Isolating an individual clone by screening libraries: general methods of preparing gene libraries, not provided for in other subgroups
C40B 40/10
Libraries containing only organic compounds: libraries containing peptides or polypeptides, or derivatives thereof
C12N
Microorganisms or enzymes

Family

PCT/EP2011/002843Parent of this division · filed 9 Jun 2011
US 2017/0369889 A1Pre-grant publication · published 28 Dec 2017
Application13/703,123Parent of this division
US 2013/0267021 A1Published application
US 11,597,937 B2This patent

From the US filing history. Foreign family members aren’t listed.