US 8,709,798 B2 · Nucleic acids for cloning and expressing multiprotein complexes

Abstract

The present invention relates to a nucleic acid containing at least one homing endonuclease site (HE) and at least one restriction enzyme site (X) wherein the HE and X sites are selected such that HE and X result in compatible cohesive ends when cut by the homing endonuclease and restriction enzyme, respectively, and the ligation product of HE and X cohesive ends can neither be cleaved by the homing endonuclease nor by the restriction enzyme. Further subject-matter of the present invention relates to a vector comprising the nucleic acid of the present invention, host cells containing the nucleic acid and/or the vector, a kit for cloning and/or expression of multiprotein complexes making use of the vector and the host cells, a method for producing a vector containing multiple expression cassettes, and a method for producing multiprotein complexes. The invention also relates to a methods of assembling multiple single vectors (“vector entities”) into fusion vectors and to method of disassembling a fusion vector containing multiple of such vector entities into single vectors. The invention is also directed to fusion vectors containing multiple vector entities.

Claims20

Dependent claims

A nucleic acid containing at least one homing endonuclease (HE) site and at least one restriction enzyme (X) site wherein the HE and X sites are selected such that a ligation product having compatible cohesive ends results when the nucleic acid is cut by a homing endonuclease and a restriction enzyme, respectively, and the ligation product having compatible cohesive ends can neither be cleaved by the homing endonuclease nor the restriction enzyme, the nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO:
50, SEQ ID NO:
51, SEQ ID NO:
52 and SEQ ID NO:
53.

2 more elements

A method for producing a vector containing multiple expression cassettes comprising the steps of:

(a) providing a first vector comprising a nucleic acid containing at least one homing endonuclease (HE) site and at least one restriction enzyme (X) site wherein the HE and X sites are selected such that a first ligation product having compatible cohesive ends results when the first vector is cut by a homing endonuclease and a restriction enzyme, respectively, and the first ligation product having compatible cohesive ends can neither be cleaved by the homing endonuclease nor the restriction enzyme;
(b) inserting one or more genes between the HE site and the X site of the first vector;
(c) providing a second vector comprising a second nucleic acid containing at least one homing endonuclease (HE) site and at least one restriction enzyme (X) site wherein the HE and X sites are selected such that a second ligation product having compatible cohesive ends results when the second vector is cut by a homing endonuclease and a restriction enzyme, respectively, and the second ligation product of HE and X cohesive ends can neither be cleaved by the homing endonuclease nor the restriction enzyme;
(d) inserting one or more genes between the HE site and the X site of the second vector;
(e) cleaving the first vector with the homing endonuclease specific for site HE and with the restriction enzyme specific for site X yielding a fragment containing the at least one gene flanked by the cleaved HE and X site;
(f) cleaving the second vector with the homing endonuclease specific for site HE;
(g) ligating the fragment obtained in step (e) into the cleaved second vector obtained in step (f);
and optionally
(h) repeating steps (a) to (g) with one or more vector(s) generating the vector containing multiple expression cassettes.

6 more elements

Dependencies are inferred from the claim text.

Nucleic acids for cloning and expressing multiprotein complexes

Life of this patent

Nucleic acids for cloning and expressing multiprotein complexes

Life of this patent

Abstract

Claims20