The present invention is related to a nucleic acid molecule, which is also referred to as third nucleic acid molecule, wherein the third nucleic acid molecule comprises (1) a nucleic acid molecule comprising the following elements: (a) optionally, a first part of a genome of a virus; (b) a nucleotide sequence, preferably a genomic nucleotide sequence, or a transcription unit; (c) a regulatory nucleic acid sequence which has a regulatory activity in a prokaryote; (d) exactly one site-specific recombination site; (e) a nucleotide sequence providing for a negative selection marker; (f) a bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for conditional replication and (ii) a nucleotide sequence providing for a positive selection marker; (g) optionally a first restriction site; or (2) a nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO: 6; or (3) a nucleic acid molecule identical or similar to the nucleic acid molecule contained in the organism deposited with the DSMZ under the Budapest treaty under accession number DSM 23754, wherein preferably the nucleic acid molecule contained in the organism is a heterologous nucleic acid molecule; wherein the third nucleic acid molecule is either a linear or a circular molecule.

The invention relates to the production and use of Cas-encoding sequences and vectors comprising these. Aspects of the invention provide products, vectors, delivery vehicles, uses and methods for producing Cas-encoding sequences in bacterial or archaeal cells.

The invention pertains to a method for producing a plant shoot, wherein said method comprises the step of locally contacting a plant tissue comprising a sequence encoding at least one regeneration factor under the control of an inducible promoter with a inductive hydrogel. The invention further pertains to an inductive hydrogel and the use of said inductive hydrogel for inducing regeneration.

Provided herein are methods for the production of a vector with a size of at least 16 kb from bacterial cells comprising the consecutive steps of a) obtaining bacterial cells comprising a vector with a size of at least 16 kb, comprising an inducible origin of replication, b) inoculating culture medium with the bacterial cells comprising the vector, c) culturing the bacterial cells in the culture medium, d) adding one or more inducers of said inducible origin of replication to the culture medium when the bacterial culture has reached an optical density at 600 nm (OD600) of at least 20, e) further culturing the bacterial cells in the culture medium, f) optionally separating the bacterial cells from the culture medium, and g) recovering the plasmid from the bacterial cells. Also provided herein are vectors with a size of at least 16 kb comprising an inducible origin of replication for use in such methods.

A vector for gene expression comprises a promoter polynucleotide sequence, a polynucleotide sequence encoding a first detectable protein in a first reading frame, a polynucleotide sequence adapted for insertion of a test sequence, a nonsense-mediated decay polynucleotide sequence, a start codon that is in a second reading frame, a polynucleotide sequence encoding a second detectable protein in the first reading frame. A polynucleotide sequence encoding a third detectable protein is optionally present. The second reading frame may be one or two reading frames removed from the first reading frame. The polynucleotide sequence encoding a third detectable protein may be flanked on the 5 and 3 ends by recombination sequences. A method of detectably inducing expression of a gene utilizes the vector.