TNT cloning system

1. A cloning system, comprising:

I. a first family of double-stranded destination vectors comprising at least two first family destination vectors 1A and 2, wherein each of the destination vectors in the first family comprises the same first type IIS restriction enzyme site, the same second type IIS restriction enzyme site, and a selectable marker gene, wherein each destination vector comprises at least three signature elements, wherein said three signature elements are different from one another, wherein each of said three signature elements consists of 3-nucleotides, and wherein each first family destination vector comprises, from 5′ to 3′ in the sense strand:

a. the forward sequence of the first type IIS restriction enzyme site;

b. the forward sequence of a first signature element;

c. the reverse complement sequence of the second type IIS restriction enzyme site;

d. the selectable marker gene;

e. the forward sequence of the second type IIS restriction enzyme site;

f. the forward sequence of a second signature element; and

g. the reverse complement sequence of the first type IIS restriction enzyme site;

wherein said first family destination vectors 1A and 2 are further organized as follows:

i. said first family destination vector 1A further comprises, from 5′ to 3′ on the sense strand, the forward sequence of a third signature element between (f) the forward sequence of the second signature element and (g) the reverse complement sequence of the first type IIS restriction enzyme site; and

ii. said first family destination vector 2 further comprises, from 5′ to 3′ on the sense strand, the forward sequence of said third signature element between (a) the forward sequence of the first type IIS restriction enzyme site and (b) the forward sequence of the first signature element;

II. a second family of double-stranded destination vectors comprising at least one second family destination vector 1A, each vector in said second family comprising the same first and second type IIS restriction enzyme sites as in the first family of destination vectors, and at least three signature elements which are the same signature elements as in the first family of destination vectors, and further comprising a selectable marker gene that can be the same or different from the selectable marker gene of said first family destination vectors, and wherein each second family destination vector comprises, from 5′ to 3′ in the sense strand:

a. the forward sequence of the second type IIS restriction enzyme site;

b. the forward sequence of the first signature element;

c. the reverse complement sequence of the first type IIS restriction enzyme site;

d. the selectable marker gene;

e. the forward sequence of the first type IIS restriction enzyme site;

f. the forward sequence of said second signature element; and

g. the reverse complement sequence of the second type IIS restriction enzyme site;

 wherein said second family destination vector 1A further comprises, from 5′ to 3′ on the sense strand, the forward sequence of the third signature element between (f) the forward sequence of the second signature element and (g) the reverse complement sequence of the second type IIS restriction enzyme site;

III. a destination vector B which further comprises, from 5′ to 3′ on the sense strand, the forward sequence of said third signature element between (a) the forward sequence of the first type IIS restriction enzyme site and (b) the forward sequence of the first signature element; and the forward sequence of a fourth signature element between (f) the forward sequence of the second signature element and (g) the reverse complement sequence of the first type IIS restriction enzyme site, wherein the fourth signature element consists of 3-nucleotides and wherein the fourth signature element is different from the first, second, and third signature elements; and

IV. a destination vector C which further comprises, from 5′ to 3′ on the sense strand, the forward sequence of said fourth signature element between (a) the forward sequence of the first type IIS restriction enzyme site and (b) the forward sequence of the first signature element.

2. The cloning system of claim 1 , wherein said second family of destination vectors further comprises a second family destination vector 2 which further comprises, from 5′ to 3′ on the sense strand, the forward sequence of said third signature element between (a) the forward sequence of the second type IIS restriction enzyme site and (b) the forward sequence of the first signature element.

3. The cloning system of claim 1 , wherein said second family of destination vectors further comprises the following second family destination vectors:

V. a destination vector B which further comprises, from 5′ to 3′ on the sense strand, the forward sequence of said third signature element between (a) the forward sequence of the second type IIS restriction enzyme site and (b) the forward sequence of the first signature element; and the forward sequence of said fourth signature element between (f) the forward sequence of the second signature element and (g) the reverse complement sequence of the second type IIS restriction enzyme site; and

VI. a destination vector C which further comprises, from 5′ to 3′ on the sense strand, the forward sequence of said fourth signature element between (a) the forward sequence of the second type IIS restriction enzyme site and (b) the forward sequence of the first signature element.

4. The cloning system of claim 1 , wherein each member of said first and second families of destination vectors further comprises a second selectable marker which is the same within each family, but which differs between the first and second families.

5. The cloning system of claim 4 , wherein said second selectable markers for said first and second families are antibiotic resistance genes.

6. The cloning system of claim 1 , wherein the first and second type IIS restriction enzyme sites are selected from restriction enzyme recognition sites for Enterobacter aerogenes I (EarI), Lysobacter gummosus RFLI (LguI), Bacillus coagulans 5I (Bco5I), Bacillus coagulans 116I (Bco116I), Bacillus coagulans KI (BcoKI), Bacillus stearothermophilus XI (BsaXI), Bacillus stearothermophilus ZI (BseZI), Bacillus stearothermophilus 6I (Bst6I), Bacillus stearothermophilus IMI (BssIMI), Bacillus sphaericus QI (BspQI), Bacillus stearothermophilus IMI (BssIMI), Bacillus subtilis 6I (Bsu6I), Bacillus atrophaeus HI (BatHI), Enterobacter amnigenus RFL1104 (Eam1104I), Klebsiella pneumoniae NIH30III (KpnNIH30III), Planococcus citreus SI (PciSI), Rhizobium leguminosarum AI (RleAI), Saccharopolyspora species I (SapI), Staphylococcus intermedius I (SimI), and Vibrio parahaemolyticus K32I (VpaK32I).

7. The cloning system of claim 6 , wherein the first and second type IIS restriction enzyme sites are selected from Enterobacter aerogenes I (EarI) and Lysobacter gummosus RFLI (LguI).

8. The cloning system of claim 1 , further comprising a ligation buffer comprising a polyglycol polymer.

9. The cloning system of claim 8 , wherein said ligation buffer further comprises 20-80 mM Tris-HCl, (pH 7-8); 0.1-4.0 mM dithiothreitol (DTT); 1-20 mM MgCl 2 ; 0.1-2.0 mM ATP; and 0.1-4.0% poly(propylene glycol) (PPG).

10. The cloning system of claim 1 , wherein said first family of destination vectors further comprises at least one vector for antisense expression of a nucleic acid, wherein said at least one vector for antisense expression is selected from:

VII. a first family destination vector 1A-R comprising, from 5′ to 3′ in the sense strand:

the forward sequence of the first type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 5′ end of the first signature element;

the forward sequence of said first signature element;

the reverse complement sequence of the second signature element;

the reverse complement sequence of the second type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 3′ end of the reverse complement sequence of said second signature element;

the selectable marker gene;

the forward sequence of the second type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 5′ end of the first reverse complement sequence of the first signature element;

the reverse complement sequence of the first signature element;

the forward sequence of the third signature element; and

the reverse complement sequence of the first type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 3′ end of a fourth signature element;

VIII. a first family destination vector 2-R which comprises, from 5′ to 3′ in the sense strand:

the forward sequence of the first type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 5′ end of the third signature element;

the forward sequence of said third signature element;

the reverse complement sequence of the second signature element;

the reverse complement sequence of the second type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 3′ end of the reverse complement sequence of said second signature element;

the selectable marker gene;

the forward sequence of the second type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 5′ end of the first reverse complement sequence of the first signature element;

the reverse complement sequence of the first signature element;

the forward sequence of the second signature element; and

the reverse complement sequence of the first type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 3′ end of the second signature element;

IX. a first family destination vector B-R which comprises, from 5′ to 3′ in the sense strand:

the forward sequence of the first type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 5′ end of the third signature element;

the forward sequence of said third signature element;

the reverse complement sequence of the second signature element;

the reverse complement sequence of the second type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 3′ end of the reverse complement sequence of said second signature element;

the selectable marker gene;

the forward sequence of the second type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 5′ end of the first reverse complement sequence of the first signature element;

the reverse complement sequence of the first signature element;

the forward sequence of the fourth signature element; and

the reverse complement sequence of the first type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 3′ end of the fourth signature element; and

X. a first family destination vector C-R which comprises, from 5′ to 3′ in the sense strand:

the forward sequence of the first type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 5′ end of the fourth signature element;

the forward sequence of said fourth signature element;

the reverse complement sequence of the second signature element;

the reverse complement sequence of the second type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 3′ end of the reverse complement sequence of said second signature element;

the selectable marker gene;

the forward sequence of the second type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 5′ end of the first reverse complement sequence of the first signature element;

the reverse complement sequence of the first signature element;

the forward sequence of the second signature element; and

the reverse complement sequence of the first type IIS restriction enzyme site, with a restriction enzyme cleavage site at the 3′ end of the second signature element.