# US 2013/0157308 A1 · Bidirectional promoter reporter vector for the analysis of dual regulatory elements

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- Number: US 2013/0157308 A1
- Document ID: US20130157308A1
- Status: Published application
- Kind: A1
- Application number: 13/713,622
- Filed: 2012-12-13
- Published: 2013-06-20
- Earliest claimed: 2011-12-15
- Assignees: University of South Carolina
- Inventors: David Reisman; Amanda Polson
- Web page: https://patentgrep.com/patent/US20130157308A1

## CPC

- C12N 15/85: for animal cells
- C12N 2830/205: bidirectional
- C12N 2830/85: mammalian
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A bidirectional expression vector is described that can be utilized to determine the existence and characteristics of bidirectional promoters. The bidirectional expression vector includes two different reporter genes in a head to head (5′ to 5′) arrangement. In addition, the bidirectional expression vector can include a polylinker region located between the heads of the two reporter genes that provides multiple cloning sites for nonexclusive examination of polynucleotide sequences. The vector can also include a splicing site and drug resistance. The bidirectional expression vector can be used to examine a polynucleotide sequence for the presence of divergent regulator regions and, following determination of a bidirectional promoter, can be utilized to further elucidate characteristics of the bidirectional promoter.

## Claims

### Claim 1

A bidirectional expression vector comprising a first reporter gene and a second reporter gene, the first and second reporter genes being arranged in a head to head (5′ to 5′) arrangement, the bidirectional expression vector including a polylinker region between the heads of the first and second reporter genes, the bidirectional expression vector including multiple cloning sites within the polylinker region.

### Claim 2 (depends on 1)

The bidirectional expression vector of claim 1, wherein the first reporter gene is a renilla gene.

### Claim 3 (depends on 1)

The bidirectional expression vector of claim 1, wherein the second reporter gene is a luciferase gene.

### Claim 4 (depends on 1)

The bidirectional expression vector of claim 1, the polylinker region including more than one copy of at least one of the restriction sites.

### Claim 6 (depends on 1)

The bidirectional expression vector of claim 1, the polylinker region including the following restriction sites: SmaI, XmaI, KpnI, SmaI, XmaI, and PstI, the restriction sites being inserted in the order as provided.

### Claim 7 (depends on 1)

The bidirectional expression vector of claim 1, the polylinker region including at least one of each of SmaI, KpnI, SacI, SalI, XhoI, and BgII as restriction sites.

### Claim 8 (depends on 1)

The bidirectional expression vector of claim 1, further including an antibiotic resistance gene.

### Claim 9 (depends on 8)

The bidirectional expression vector of claim 8, wherein the antibiotic resistance gene is an ampicillin resistance gene.

### Claim 10 (depends on 1)

The bidirectional expression vector of claim 1, further comprising a splicing signal.

### Claim 11 (depends on 1)

The bidirectional expression vector of claim 1, further comprising a polyadenylation signal.

### Claim 12 (depends on 11)

The bidirectional expression vector of claim 11, further comprising a first polyadenylation signal downstream of the first reporter gene and a second polyadenylation signal downstream of the second reporter gene.

### Claim 13 (depends on 1)

A host cell comprising the bidirectional expression vector of claim 1.

### Claim 14 (depends on 13)

The host cell of claim 13, wherein the host cell is a mammalian cell.

### Claim 15 (depends on 14)

The host cell of claim 14, wherein the host cell is a human cell or a murine cell.

### Claim 16 (depends on 1)

A kit comprising the bidirectional expression vector of claim 1.

### Claim 17

A method for the production of a first expression product of a first reporter gene and a second expression product of a second reporter gene from a bidirectional vector, the method including:
cloning a polynucleotide into a cloning site of the bidirectional vector, the bidirectional vector including the first reporter gene and the second reporter gene in a head to head (5′ to 5′) arrangement and a polylinker region between the heads of the first and second reporter gene, the polylinker region including the cloning site and further including at least one additional cloning site;
transfecting a host cell with the bidirectional vector; and
culturing the host cell.

### Claim 18 (depends on 17)

The method according to claim 17, further comprising determining the presence or quantity of at least one of the first and second expression products.

### Claim 19 (depends on 17)

The method according to claim 17, further comprising incorporating a mutation into the polynucleotide.

### Claim 20 (depends on 17)

The method according to claim 17, further comprising deleting a segment of the polynucleotide.