# US 6,294,372 B1 · Replication genes and gene products from small cryptic plasmids and methods for constructing controlled-replication plasmid vectors

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- Number: US 6,294,372 B1
- Document ID: US6294372B1
- Status: Granted
- Kind: B1
- Application number: 09/042,071
- Filed: 1998-03-13
- Granted: 2001-09-25
- Earliest claimed: 1997-03-14
- Assignees: University of Victoria Innovation & Dev. Corp.
- Inventors: Jàn Burian; William W. Kay
- Web page: https://patentgrep.com/patent/US6294372B1

## CPC

- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C07K 14/245: Escherichia (G)
- C07K 2319/00: Fusion polypeptide
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The replication genes of small cryptic plasmids are isolated and used to construct controlled-replication plasmid vectors with the wide range of copy numbers controlled by defined helper plasmids. Controlled-replication vectors (RAMP vectors) can reach very high level of plasmid replication, which is not lethal to host unlike runaway replication vectors.

## Claims

### Claim 1

An isolated nucleic acid molecule which encodes a repA protein, wherein said nucleic acid molecule is selected from the group consisting of:
(a) a nucleic acid molecule which encodes a protein comprising the amino acid sequence of RepA wherein said protein is encoded by nucleotides 913 to 1386 of Sequence I.D. No: 19;
(b) an isolated nucleic acid molecule that detectably hybridizes to the nucleic acid molecule of (a) or its complementary strand under conditions of 5×SSPE, 0.5% SDS, 1×Denhardt's, at 65° C. or equivalent salt and temperature conditions;
(c) an isolated nucleic acid molecule that encodes a protein that has greater than 75% sequence homology to the protein encoded by the nucleic acid molecule according to (a); and
(d) a nucleic acid molecule that, due to degeneracy of the genetic code, encodes a protein encoded by the nucleic acid molecule according to (b) or (c).

### Claim 2 (depends on 1)

A vector that contains a nucleic acid molecule according to claim 1.

### Claim 3 (depends on 2)

The vector according to claim 2, further comprising a heterologous nucleic acid molecule.

### Claim 4 (depends on 2)

A host cell that contains a vector according to claim 2.

### Claim 5 (depends on 1)

A vector that directs the expression of a nucleic acid molecule according to claim 1.

### Claim 6 (depends on 5)

The vector according to claim 5, further comprising a heterologous nucleic acid molecule.

### Claim 7 (depends on 5)

A host cell that contains a vector according to claim 5.

### Claim 8 (depends on 1)

A vector which contains a promoter recognition sequence of the protein encoded by the isolated nucleic acid molecule according to claim 1.

### Claim 9

A vector designated pKL1.

### Claim 10 (depends on 8, 9)

The vector according to any one of claims 9 or 8, further comprising a heterologous nucleic acid molecule.

### Claim 11 (depends on 8, 9)

A host cell that contains a vector according to any one of claim 9 or 8.