# US 10,865,407 B2 · Cloning method

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,865,407 B2
- Document ID: US10865407B2
- Status: Granted
- Kind: B2
- Application number: 15/648,983
- Filed: 2017-07-13
- Published: 2017-11-02
- Granted: 2020-12-15
- Earliest claimed: 2012-03-27
- Assignees: Dsm IP Assets B.V.
- Applicants: Dsm IP Assets B.V.
- Inventors: Johannes Andries Roubos; Herman Jan Pel; Bernard Meijrink
- Web page: https://patentgrep.com/patent/US10865407B2

## CPC

- C12N 15/1024: {In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair}
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- C12N 2800/40: Systems of functionally co-operating vectors
- C12N 2800/50: Vectors for producing vectors
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a method based on the use of restriction enzyme digestion and ligation via the cleavage sites, thereby to prepare two or more standardized expression cassettes.

## Claims

### Claim 1

A system for producing a nucleic acid construct of interest incorporated at a target locus, said system comprising:
I) a system for preparing two or more standardized expression cassettes, said system comprising:
a) two or more sets of element sequences,
each set of element sequences together comprising at least one functional expression cassette,
each element sequence being flanked on both sides by a type Ils restriction endonuclease cleavage site followed by the recognition site thereof,
the type IIs restriction endonuclease recognition sites and cleavage sites being selected so that the sets of element sequences may be assembled into a functional expression cassette;
b) at least two backbone entry vectors,
each backbone entry vector comprising in this order (i) a restriction enzyme cleavage site with its recognition site and a first connector sequence (LF); (ii) a vector backbone comprising a selectable marker gene; and (iii) a second connector sequence (RF) and a restriction enzyme recognition site with its cleavage sequence, and; (iv) optionally, an insert between the recognition sites of (i) and (iii),
the connector sequences, RF and LF, on any backbone entry vector being selected so that they can assemble with a LF or RF connector sequence respectively on the same or a different backbone entry vector; and
II) at least two double-stranded integration sequences, one of which is capable of recombination with a first expression cassette comprising at least one of the two or more sets of element sequences of I) a) and a sequence flanking the target locus, and the second of which is capable of recombination with a second expression cassette comprising at least one of the two or more sets of element sequences of I) a) and a sequence flanking the other side of the target locus.

### Claim 2 (depends on 1)

The system according to claim 1, wherein the at least two backbone entry vectors comprise, in this order (i) a type II restriction enzyme recognition site followed by the cleavage site thereof and a first at least 25-basepair connector sequence (LF); (ii) a vector backbone comprising a selectable marker gene; and (iii) a second at least 25-basepair connector sequence (RF) and a further cleavage site of a type II restriction enzyme followed by the recognition site of said cleavage site, and; (iv) optionally, an insert between the recognition sites of (i) and (iii).

### Claim 3 (depends on 1)

The system according to claim 1, wherein the integration sequences are capable of mediating integration via homologous recombination or site-specific recombination.

### Claim 4 (depends on 1)

The system according to claim 1, wherein the integration sequences comprise additional sequences for recombination with a second target locus, optionally a locus in a host cell of species different than the first target locus.

### Claim 5 (depends on 4)

The system according to claim 4, wherein the integration sequences comprise site-specific recombination sequences for recombination with the second target locus, optionally at a target locus in a host cell species different than the host cell species for the first target locus.

### Claim 6 (depends on 1)

The system according to claim 1, wherein at least two of the sets of element sequences comprise a promoter element, an open reading frame element, and a terminator element.

### Claim 7 (depends on 6)

The system according to claim 6, wherein at least one of the sets of element sequences comprises an open reading frame element encoding a marker.

### Claim 8 (depends on 7)

The system according to claim 7, wherein the at least one set of element sequences comprising an open reading frame element encoding a marker is flanked by at least two site-specific recombination sites.