Patent US4153838A successfully refreshed from Google Patents.
Patent US4495280A successfully refreshed from Google Patents.
US4495280A
Expired / Lapsed
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Cloned high signal strength promoters
Assignee: Leland Stanford Junior University
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Filing: 1981-05-20
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Granted: 1985-01-22
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Expires: 2001-05-20
Abstract
Abstract Method for preparing high signal strength promoters and terminators and DNA compositions employing such promoters and terminators. T5 phage is cleaved to provide for DNA sequences having intact promoters. These promoters are inserted into vectors separated from a balanced terminator by a gene of interest and the terminator is desirably followed by a marker allowing for selection of transformants. High efficiencies in transcription of DNA can be achieved with the highly active T5 promoters. The promoters and terminators are used in hybrid DNA for efficient expression of structural genes and transcription to provide RNA sequences.
Inventors
- 👤 Hermann G. Bujard
- 👤 Annie C. Y. Chang
- 👤 Stanley N. Cohen
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 07:33
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/70
Claims Analysis
Total Claims: 18
Claim 1
Independent Claim
122 words
A linear DNA sequence having proximal to one end a strong T5 phage promoter, proximal to the other end a strong transcriptional terminator balanced with said strong T5 promoter, and having intermediate said promoter and terminator at least one of (1) a marker for selection adjacent to said terminator or (2) a replication system foreign to T5, wherein the direction of said promoter is away from said terminator and said marker is expressed at a frequency of less than about one-fourth the frequency of a structural gene, when said structural gene is inserted between said promoter and terminator, so as to be under the transcriptional control of said promoter and to bridge said linear DNA sequence to provide a circular DNA sequence.
Claim 2
Dependent Claim
122 words
A linear DNA sequence having proximal to one end a strong T5 phage promoter, proximal to the other end a strong transcriptional terminator balanced with said strong T5 promoter, and having intermediate said promoter and terminator at least one of (1) a marker for selection adjacent to said terminator or (2) a replication system foreign to T5, wherein the direction of said promoter is away from said terminator and said marker is expressed at a frequency of less than about one-fourth the frequency of a structural gene, when said structural gene is inserted between said promoter and terminator, so as to be under the transcriptional control of said promoter and to bridge said linear DNA sequence to provide a circular DNA sequence.
Claim 3
Dependent Claim
28 words
A linear DNA sequence according to claim 1, having intermediate said other end and said strong terminator at least one stop codon in at least one reading frame.
Claim 4
Dependent Claim
22 words
A linear DNA sequence according to claim 2, having a plurality of stop codons with at least one in each reading frame.
Claim 5
Dependent Claim
22 words
A linear DNA sequence according to any of claims 1, 2 or 3, wherein said marker is a gene imparting biocidal resistance.
Claim 6
Dependent Claim
30 words
A linear DNA sequence according to any of claims 1, 2 or 3, wherein said marker is a DNA sequence having at least one gene in a metabolic synthetic pathway.
Claim 7
Dependent Claim
22 words
A linear DNA sequence according to any of claims 1, 2 or 3, having a marker intermediate said promoter and said terminator.
Claim 8
Dependent Claim
14 words
A linear DNA sequence according to claim 6, wherein said marker provides biocidal resistance.
Claim 9
Dependent Claim
24 words
A linear DNA sequence according to claim 6, wherein said marker has at least one gene for an enzyme in a metabolic synthetic pathway.
Claim 10
Dependent Claim
21 words
A linear DNA sequence according to any of claims 1, 2 or 3, wherein said replication system is for a prokaryote.
Claim 11
Dependent Claim
21 words
A linear DNA sequence according to any of claims 1, 2 or 3, wherein said replication system is for a eukaryote.
Claim 12
Independent Claim
117 words
A method for determining the strength of a promoter which comprises: inserting said promoter into a linear DNA sequence having in the downstream direction for expression proximal to one end; a gene allowing for detection of expression; a transcriptional terminator of known strength; a marker allowing for determination of expression; and a replication system recognized by a predetermined host; whereby a circular DNA sequence is obtained; transforming said host with said circular DNA sequence; growing said host in nutrient medium under conditions allowing for determination of the extent of expression of said gene and said marker; and determining the strength of said promoter is determined by the relative degree of transcription of said gene and said marker.
Claim 13
Independent Claim
117 words
A method for determining the strength of a promoter which comprises: inserting said promoter into a linear DNA sequence having in the downstream direction for expression proximal to one end; a gene allowing for detection of expression; a transcriptional terminator of known strength; a marker allowing for determination of expression; and a replication system recognized by a predetermined host; whereby a circular DNA sequence is obtained; transforming said host with said circular DNA sequence; growing said host in nutrient medium under conditions allowing for determination of the extent of expression of said gene and said marker; and determining the strength of said promoter is determined by the relative degree of transcription of said gene and said marker.
Claim 14
Dependent Claim
16 words
A method according to claim 11, wherein said host is auxotrophic and said gene provides prototrophy.
Claim 15
Independent Claim
16 words
A method according to any of claims 11 or 12, wherein said marker provides biocidal resistance.
Claim 16
Dependent Claim
29 words
A method according to claim 13, wherein intermediate said gene and said terminator are a plurality of stop codons, with at least one stop codon in each reading frame.
Claim 17
Dependent Claim
81 words
A circular DNA sequence having in downstream order of transcription a strong T5 phage promoter, a structural gene foreign to T5 phage under transcriptional control of said promoter, a transcriptional terminator which is balanced with said promoter and a replication system and having a marker for selection downstream from said terminator, wherein said marker is expressed at a frequency of less than about one-fourth the frequency of which the structural gene is expressed and is under transcriptional control of said promoter.
Claim 18
Dependent Claim
81 words
A circular DNA sequence having in downstream order of transcription a strong T5 phage promoter, a structural gene foreign to T5 phage under transcriptional control of said promoter, a transcriptional terminator which is balanced with said promoter and a replication system and having a marker for selection downstream from said terminator, wherein said marker is expressed at a frequency of less than about one-fourth the frequency of which the structural gene is expressed and is under transcriptional control of said promoter.
References Cited (Backward Citations)
No citation records indexed.
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-01-22 | 1988-07-23 | 1989-01-22 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-01-22 | 1992-07-23 | 1993-01-22 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-01-22 | 1996-07-23 | 1997-01-22 | $7700.00 | Window Closed / Lapsed |