Cloning vectors and vector components

• US 6,709,861 B2
• Filed: 11/15/2001
• Issued: 03/23/2004
• Est. Priority Date: 11/17/2000
• Status: Active Grant

• Current Assignee: Lucigen Corporation (3M Company)
• Original Assignee: Lucigen Corporation (3M Company)
• Inventors: Mead, David Alan, Godiska, Ronald
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): SULLIVAN, DANIEL M
• Application Number:
  US10/001,052
• Publication Number:
  US 20040038401A1
• Time in Patent Office: 859 Days
• Field of Search: 435/320.1, 536/23.1
• US Class Current: 435/320.1
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

The present invention relates to systems, methods, and compositions for cloning and sequencing insert nucleic acid sequences. In particular, the present invention provides vectors and vector components configured for multiplex cloning, multiplex sequencing, and fixed orientation cloning. The present invention also provides vectors and vector components that allow insert sequences that are deleterious to a host cell to be successfully cloned.

Claims

1. A composition comprising a vector component, wherein said vector component comprises:

i) first and second free ends;

ii) a selectable marker region,

iii) an origin of replication

iv) a first transcriptional terminator between said first free end and said selectable marker region,

v) a second transcriptional terminator between said second free end and said origin of replication, and

vi) a third transcriptional terminator between said origin of replication and said selectable marker region,

and wherein said vector component is configured to form a circular recombinant vector when said first and second free ends are attached to an insert sequence.

2. The composition of claim 1, wherein said first transcriptional terminator is configured to terminate RNA transcripts entering said selectable marker region from said first free end.

3. The composition of claim 1, wherein said second transcriptional terminator is configured to terminate RNA transcripts entering said selectable marker region from said second free end.

4. The composition of claim 1, wherein said third transcriptional terminator is configured to terminate RNA transcripts encoding at least one selectable marker sequence in said selectable marker region.

5. The composition of claim 1, wherein said vector component comprises a first non-promoter sequence between said first free end and said selectable marker region, and a second non-promoter sequence between said second free end and said selectable marker region, wherein each of said first and second non-promoter sequences are unable to serve as an operable promoter in a bacterial host cell.

6. The composition of claim 1, wherein said first and second free ends are dephosphorylated.

7. A composition comprising a circular vector, wherein said circular vector comprises:

a) a nucleic acid sequence comprising;

i) first and second ends, ii) a selectable marker region, iii) a first transcriptional terminator between said first end and said selectable marker region, and iv) a second transcriptional terminator between said second end and said selectable marker region; and

b) an insert sequence, wherein said insert sequence is attached to said first and second ends, and wherein said insert sequence comprises a base composition that is at least 65% A and T.

8. The composition of claim 7, wherein said insert sequence encodes a peptide that is deleterious to a host cell.

9. The composition of claim 7, wherein said insert sequence contains transcriptional promoters or regions that behave as promoters in bacteria.

10. The composition of claim 7, wherein said first transcriptional terminator is configured to terminate RNA transcripts entering said selectable marker region from said first end.

11. The composition of claim 7, wherein said second transcriptional terminator is configured to terminate RNA transcripts entering said selectable marker region from said second end.

12. The composition of claim 7, wherein said selectable marker region comprises a transcriptional terminator configured to terminate RNA transcripts encoding at least one selectable marker sequence in said selectable marker region.

13. The composition of claim 7, wherein said nucleic acid sequence comprises a first non-promoter sequence between said first end and said selectable marker region, and a second non-promoter sequence between said second end and said selectable marker region, wherein each of said first and second non-promoter sequences are unable to serve as an operable promoter in a bacterial host cell.

14. A composition comprising a vector component, wherein said vector component comprises:

i) first and second free ends;

ii) a selectable marker region comprising a selectable marker sequence, wherein said selectable marker sequence comprises a first promoter,

iii) a first transcriptional terminator between said first free end and said selectable marker region;

iv) a second transcriptional terminator between said second free end and said selectable marker region, and

v) a third transcriptional terminator between said second free end and said selectable marker region,

and wherein no other promoters are present in said vector component except said first promoter and said vector component is configured to form a circular recombinant vector when said first and second free ends are attached to an insert sequence.

15. The composition of claim 14, wherein said first and second free ends are dephosphorylated.