Unique modular vector design

Abstract

The current invention provides a modular vector system that enables the insertion of gene expression cassettes in a recursive directional stacking fashion by rare restriction sites which requires only one type of vector. The invention also provides DNA molecules, compositions, and transgenic organisms, plants, plant tissues, plant seeds, and cells comprising recombined restriction sites for rare restriction enzymes.

1 . A recombinant DNA molecule comprising SEQ ID NO:1, 2, 3, or 4. 
     
     
         2 . A transgenic organism comprising SEQ ID NO:1, 2, 3, or 4. 
     
     
         3 . A transgenic plant or plant part, comprising the recombinant DNA molecule of  claim 1 . 
     
     
         4 . The transgenic plant or plant part of  claim 3 , wherein said plant part comprises a cell, a seed, a boll, a leaf, a flower, a stem, a root, or any portion thereof; or a non-regenerable portion of said seed, boll, leaf, flower, stem, or root or any portion thereof. 
     
     
         5 . A vector comprising the recombinant DNA molecule of  claim 1 . 
     
     
         6 . The recombinant DNA molecule of  claim 1 , wherein SEQ ID NO:1, 2, 3, or 4 are located between two gene expression cassettes, or generated through a recombination event. 
     
     
         7 . A recombinant DNA molecule comprising:
 (a) a first arrangement of rare restriction enzyme recognition sites, having a first recognition site for a first rare restriction enzyme producing an overhang, a second recognition site for a second rare restriction enzyme producing a blunt end, and a third recognition site for a third rare restriction enzyme producing an overhang; and   (b) a second arrangement of rare restriction enzyme recognition sites, having a fourth recognition site for a rare restriction enzyme producing a blunt end, a fifth recognition site for a rare restriction enzyme producing an overhang, and a sixth recognition site for a rare restriction enzyme producing a blunt end, wherein said first or said third recognition site is compatible with said fifth recognition site, and wherein recombination of the overhangs produced by said first or said third restriction enzyme with the overhang produced by said fifth restriction enzyme eliminates said first or said third recognition site and said fifth recognition site.   
     
     
         8 . The recombinant DNA molecule of  claim 7 , wherein said first, said third, and said fifth rare restriction enzymes are homing enzymes. 
     
     
         9 . The recombinant DNA molecule of  claim 7 ,
 wherein elimination of said first or said third recognition site and said fifth recognition site eliminates recognition sites of I-SceI and PI-PspI-RC   wherein elimination of said first or said third recognition site and said fifth recognition site eliminates recognition sites of I-SceI-RC and PI-PspI;   wherein recombination of the overhangs produced by said first or said third restriction enzyme with the overhang produced by said fifth restriction enzyme generates SEQ ID NO:1, 2, 3, or 4;   wherein the recombinant DNA further comprises at least one gene expression cassette flanked by said first and said second arrangement of rare restriction enzyme recognition sites; and/or   wherein said DNA molecule further comprises a vector.   
     
     
         10 - 13 . (canceled) 
     
     
         14 . A gene expression cassette that is flanked by said first and said second claim arrangement of rare restriction enzyme recognition sites of  claim 7 . 
     
     
         15 . A vector comprising the recombinant DNA molecule of  claim 7 . 
     
     
         16 . The vector of  claim 15 , wherein the restriction site for PI-PspI or I-SceI is in reverse complemented orientation. 
     
     
         17 . The vector of  claim 15 , wherein a second restriction site for PI-PspI or I-SceI is in reverse complemented orientation. 
     
     
         18 . A method of stacking at least two gene expression cassettes, comprising:
 (a) linearizing a first vector comprising a first gene expression cassette with a first set of two rare restriction enzymes;   (b) isolating a second gene expression cassette from a second vector with a second set of two rare restriction enzymes; and   (c) combining said second gene expression cassette from step (b) with the linearized vector from step (a) to assemble a vector comprising at least two gene expression cassettes.   
     
     
         19 . The method of  claim 18 , wherein said first vector or said second vector comprise at least two gene expression cassettes. 
     
     
         20 . The method of  claim 18 , wherein said first vector is the recombinant DNA molecule of claim  12 . 
     
     
         21 . The method of  claim 18 , wherein said second vector is the recombinant DNA molecule of claim  12 . 
     
     
         22 . The method of  claim 18 , wherein a first recognition site of said first set or said second set of two rare restriction enzymes is retained, a second recognition site of said first set or said second set of two rare restriction enzymes is lost, and whereby said second gene expression cassette reintroduces the recognition site that was lost.