# US 9,752,154 B2 · High-throughput DNA fragment assembly

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- Number: US 9,752,154 B2
- Document ID: US9752154B2
- Status: Granted
- Kind: B2
- Application number: 14/416,799
- Filed: 2013-07-23
- Published: 2015-06-25
- Granted: 2017-09-05
- Earliest claimed: 2012-07-26
- Assignees: Dow AgroSciences LLC
- Applicants: Dow Agrosciences LLC; Sandeep Kumar; Steven L. Evans; Manju Gupta
- Inventors: Sandeep Kumar; Steven L. Evans; Manju Gupta
- Web page: https://patentgrep.com/patent/US9752154B2

## CPC

- C12N 15/8209: {Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers}
- C12N 15/68: Stabilisation of the vector
- C12N 15/82: for plant cells {, e.g. plant artificial chromosomes (PACs)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This invention is related to methods and systems for vector assembly for transgenic plants. A uniform modular process is used to reduce cycle time and the methods and systems provided herein can increase cloning throughput using multiple-well plates, for example 96-well plates. In some embodiments, the methods and systems provided herein eliminate or reduce the need for sequencing confirmation because no PCR is involved in the vector assembly process.

## Claims

### Claim 1

A method for DNA assembly, comprising,
(a) providing a first DNA molecule comprising a Kozak sequence, wherein the Kozak sequence is selected from the group consisting of SEQ ID NOs: 1-43 and 64-74;
(b) providing a second DNA molecule comprising the same Kozak sequence of step (a) at one end and a six frame stop sequence at the other end, wherein the six frame stop sequence is selected from the group consisting of SEQ ID NOs: 75-80;
(c) providing a third DNA molecule comprising the same six frame stop sequence of step (b);
(d) providing a fourth DNA molecule comprising a linear vector sequence; and
(e) assembling a product vector using the DNA molecules of steps (a), (b), (c), and (d) in the presence of at least one recombinase, where the Kozak sequence and the six frame stop sequence provide junction homologies for DNA recombination and wherein the product vector has sequences in the order of the DNA molecules from steps (a), (b), (c), and (d).

### Claim 2 (depends on 1)

The method of claim 1, wherein the DNA molecules of steps (a), (b), (c), and (d) are obtained using PCR amplification or direct DNA synthesis.

### Claim 3 (depends on 1)

The method of claim 1, wherein no DNA amplification technique is used.

### Claim 4 (depends on 1)

The method of claim 1, wherein polymerase chain reaction is not used.

### Claim 5 (depends on 1)

The method of claim 1, wherein the fourth DNA molecule comprises a lethal gene.

### Claim 6 (depends on 5)

The method of claim 5, wherein the lethal gene is ccdB.

### Claim 7 (depends on 1)

The method of claim 1, wherein the same Kozak sequence of both step (a) and (b) comprises a same three-frame stop sequence at its 5′ end.

### Claim 8 (depends on 7)

The method of claim 7, wherein the three-frame stop sequence is selected from the group consisting of SEQ ID NOS 44-62.