# US 11,999,959 B2 · Binary vectors and uses of same

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

- Number: US 11,999,959 B2
- Document ID: US11999959B2
- Status: Granted
- Kind: B2
- Application number: 18/114,264
- Filed: 2023-02-26
- Published: 2023-11-16
- Granted: 2024-06-04
- Earliest claimed: 2017-06-12
- Assignees: Juan Antonio García Álvarez
- Applicants: Juan Antonio García Álvarez
- Inventors: Fabio Pasin; Juan Antonio García Álvarez; Leonor Cecilia Bedoya Rojas; Maria del Carmen Simón Mateo; Diego Vicente Orzáez Calatayud; Juan Miguel Bernabé Orts
- Web page: https://patentgrep.com/patent/US11999959B2

## CPC

- C12N 15/8205: {Agrobacterium mediated transformation}
- C12N 15/8241: {Phenotypically and genetically modified plants via recombinant DNA technology}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention relates to binary vectors based on compatible and autonomous origins, specifically based on the pBBR1 and RK2 replication origins. These binary vectors are useful for having a wide range of hosts, for their maintenance in Agrobacterium sp. and Escherichia coli, and as a new tool for plant synthetic biology as well as a flexible framework for assembly, transfer and characterization of multiple DNA elements. The binary vectors disclosed are small, preferably less than 3.8 kb in size, stable, include an origin compatible with the most commonly used binary T-DNA vectors, comply with current standards for plant synthetic biology, and allow the administration of multiple T-DNA cassettes by means of the multiplexing of the vectors. The present invention also relates to methods for transferring and expressing nucleic acid sequences using said binary vectors, and to the uses of the same.

## Claims

### Claim 1

A binary vector comprising a nucleic acid sequence comprising in this order:
(a) a T-DNA right border comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 102 and SEQ ID NO: 115; and
(b) a sequence portion connecting item (a) with the following item (c); and
(c) a bacterial terminator; and
(d) an origin of replication that is functional in at least one bacterial species; and
(e) a bacterial terminator; and
(f) a sequence portion connecting item (e) with the following item (g); and
(g) a T-DNA left border comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 116; and
wherein the bacterial terminator comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111; and
wherein the sequence of item (b) and item (f) has a size of less than 200 nucleotides.

### Claim 2 (depends on 1)

A binary vector according to claim 1, wherein the origin of replication comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107.

### Claim 3 (depends on 2)

A binary vector according to claim 2 comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 28.

### Claim 4 (depends on 1)

A method for obtaining a binary vector comprising at least one nucleotide sequence of interest, said method comprising operably linking at least one nucleotide sequence of interest to the sequence of at least one binary vector according to claim 1.

### Claim 5 (depends on 4)

A binary vector obtained from the method according to claim 4.

### Claim 6 (depends on 1)

A host cell comprising one or more binary vector(s) according to claim 1.

### Claim 7 (depends on 1)

A method for delivering at least one nucleotide sequence of interest in at least one eukaryotic cell or organism comprising:
(a) operably linking at least one nucleotide sequence of interest to the sequence of at least one binary vector according to claim 1; and
(b) contacting with or introducing into at least one eukaryotic cell or organism the item obtained from step (a); or
(c) introducing the item obtained from step (a) into a host cell, and contacting with or introducing into at least one eukaryotic cell or organism the obtained host cell.

### Claim 8 (depends on 7)

A eukaryotic cell or organism, regenerated cell, regenerated organism, progeny or seed obtainable by the method according to claim 7.

### Claim 9 (depends on 1)

A kit comprising:
(a) a binary vector according to claim 1; and
(b) instructions for using the kit.

### Claim 10 (depends on 6)

A kit comprising:
(a) the host cell according to claim 6; and
(b) instructions for using the kit.

### Claim 11 (depends on 1)

A binary vector system comprising:
(a) a binary vector according to claim 1; and
(b) one or more binary vector(s).

### Claim 12

A binary vector system comprising:
(a) a first binary vector comprising an origin of replication comprising the nucleotide sequence of SEQ ID NO: 105; and
(b) a second binary vector comprising an origin of replication comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 106 and SEQ ID NO: 107; and
wherein item (a) and item (b) further comprise a T-DNA right border comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 102 and SEQ ID NO: 115; and a T-DNA left border comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 116.

### Claim 13 (depends on 12)

A binary vector system according to claim 12 comprising:
(a) a first binary vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 28; and
(b) a second binary vector comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 24.

### Claim 14 (depends on 12)

A method for obtaining a binary vector system comprising at least one nucleotide sequence of interest, said method comprising operably linking at least one nucleotide sequence of interest to the sequence of at least one binary vector of the binary vector system according to claim 12.

### Claim 15 (depends on 14)

A binary vector system obtained from the method according to claim 14.

### Claim 16 (depends on 12)

A host cell comprising the binary vector system according to claim 12.

### Claim 17 (depends on 12)

A method for delivering at least one nucleotide sequence of interest in at least one eukaryotic cell or organism comprising:
(a) operably linking at least one nucleotide sequence of interest to the sequence of at least one binary vector of the binary vector system according to claim 12; and
(b) contacting with or introducing into at least one eukaryotic cell or organism the item obtained from step (a); or
(c) introducing the item obtained from step (a) into a host cell, and contacting with or introducing into at least one eukaryotic cell or organism the obtained host cell.

### Claim 18 (depends on 17)

A eukaryotic cell or organism, regenerated cell, regenerated organism, progeny or seed obtainable by the method according to claim 17.

### Claim 19 (depends on 12)

A kit comprising:
(a) a binary vector system according to claim 12; and
(b) instructions for using the kit.

### Claim 20 (depends on 16)

A kit comprising:
(a) the host cell according to claim 16; and
(b) instructions for using the kit.