Methods for dynamic vector assembly of dna cloning vector plasmids

Abstract

A method for using cloning vector plasmids to produce DNA molecules, such as transgenes, in a single cloning step. The transgenes can be used for the purpose of gene expression or analysis of gene expression. The plasmid cloning vectors are engineered to minimize the amount of manipulation of DNA fragment components by the end user of the vectors and the methods for their use. Transgenes produced using the invention may be used in a single organism, or in a variety of organisms including bacteria, yeast, mice, and other eukaryotes with little or no further modification.

1 . A method for constructing a transgene, comprising the steps of:
 a. providing a cloning vector plasmid with a backbone able to accept a sequential arrangement of inserts;   b. providing at least a first insert and a second insert to be included in the transgene; and   c. transferring both the first insert and the second insert into the backbone in a single reaction, wherein at least said first insert and said second insert each are arranged in a predetermined order and orientation.   
     
     
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         19 . A method of preparing one or more transgenes comprising the steps of:
 (a) providing a cloning vector comprising at least a first and a second docking point, wherein said first and said second docking points each comprise a nucleotide sequence that is recognized by at least a first and a second rare restriction enzyme, respectively, and wherein said first and said second rare restriction enzyme are different from each other;   (b) digesting said cloning vector using said first and said second rare restriction enzyme to obtain a digested vector having a 5′ end and a 3′ end;   (c) providing at least a first and a second polynucleotide insert wherein each of said first and second polynucleotide inserts comprises a 5′ insert end, a nucleotide sequence of interest and a 3′ insert end;   (d) placing said first polynucleotide insert and said second polynucleotide inserts and said digested vector together into an appropriate reaction mixture to allow annealing and ligating of nucleic acids thereby obtaining an insert-bearing vector,   wherein said first polynucleotide insert and said second polynucleotide insert and said digested vector anneal and ligate with each other in a predetermined order and orientation such that said first docking point of said cloning vector is re-formed between said 5′ end of said first polynucleotide insert and said 3′ end of said digested vector, and said second docking point of said cloning vector is re-formed between said 3′ insert end of said second insert and said 5′ end of said digested vector; and   wherein at least one additional docking point is formed between the re-formed first and re-formed second docking points, and   each additional docking point comprises a nucleotide sequence that is recognized by an additional rare restriction enzyme that is different from said first rare and said second rare restriction enzymes.   
     
     
         20 . The method of  claim 19 , wherein said at least first and second polynucleotide inserts are prepared by a method selected from the group consisting of de novo synthesis, recombineering, and PCR. 
     
     
         21 . The method of  claim 19  wherein said additional docking point is formed between the re-formed first and second docking points upon ligation of said 3′ end of said first polynucleotide insert and said 5′ end of said second polynucleotide insert. 
     
     
         22 . The method of  claim 19 , wherein said first polynucleotide insert comprises a promoter sequence and said second polynucleotide insert comprises a nucleotide sequence selected from the group consisting of an enhancer sequence, a promoter sequence, a gene of interest, a gene capable of being expressed as an mRNA an expression module, an intron, a reporter sequence, a tag sequence, a splicing signal sequence, an RNA stabilization element and a regulatory sequence. 
     
     
         23 . The method of  claim 19 , further comprising providing a third polynucleotide insert, wherein a third docking point and a fourth docking point are formed between the re-formed first and re-formed second docking points upon ligation of said first, said second, said third polynucleotide inserts and said digested vector. 
     
     
         24 . The method of  claim 23 , wherein said first polynucleotide insert comprises a promoter sequence, said second polynucleotide insert comprises an expression module sequence and said third polynucleotide insert comprises a regulatory sequence, respectively. 
     
     
         25 . The method of  claim 23 , further comprising providing a fourth polynucleotide insert, wherein a fifth docking point and a sixth docking point are formed between the re-formed first and re-formed second docking points upon ligation of said first, said second, said third, said fourth polynucleotide inserts and said digested vector. 
     
     
         26 . The method of  claim 19 , wherein said first and second docking points each comprise a nucleotide sequence that is recognized by more than one rare restriction enzyme. 
     
     
         27 . The method of  claim 19  further comprising the steps of:
 (e) digesting said insert-bearing vector to remove at least one polynucleotide insert from said insert-bearing vector thereby providing a digested insert-bearing vector;   (f) placing at least one different polynucleotide insert(s) and said digested insert-bearing vector into a reaction vessel under appropriate conditions to allow annealing and ligation of nucleic acids, wherein said different polynucleotide insert(s) and said digested vector anneal and ligate with each other in a predetermined order and orientation such that said first docking point of said cloning vector is again re-formed and at least one additional docking point is formed between the re-formed first docking point and the re-formed second docking point, and   wherein said additional docking point(s) comprises a nucleotide sequence that is recognized by an additional rare restriction enzyme that is different from said first and said second rare restriction enzymes.   
     
     
         28 . The method of  claim 27 , wherein the insert that is removed is a first promoter and said different insert is a second promoter. 
     
     
         29 . The method of  claim 19  further comprising the steps of:
 (e) digesting said insert-bearing vector to remove at least one polynucleotide insert from said insert-bearing vector thereby providing a digested insert-bearing vector;   (f) placing at least one different polynucleotide insert(s) and said digested insert-bearing vector into a reaction vessel under appropriate conditions to allow annealing and ligation of nucleic acids,   wherein said different polynucleotide insert(s) and said digested vector anneal and ligate with each other in a predetermined order and orientation such that said second docking point of said cloning vector is again re-formed and at least one additional docking point is formed between the re-formed first docking point and the re-formed second docking point, and   wherein said additional docking point(s) comprises a nucleotide sequence that is recognized by an additional rare restriction enzyme that is different from said first and said second rare restriction enzymes.   
     
     
         30 . The method of  claim 29 , wherein the insert that is removed is a first expression module and said different polynucleotide insert is a second expression module. 
     
     
         31 . The method of  claim 19 , wherein each of said first rare restriction enzyme and said second rare restriction enzyme is selected from the group consisting of Acl I, Asc I, AsiS I, BsiW I, Fse I, Mlu I, Not I, Nm I, Pac I, Pme I, Pvu I, Rsr II, Sal I, Sbf I, Sfi I, SgrA I, SnaB I, Swa I, Pl-Sce I, I-Sce I, I-Ceu I, PL-Psp I, I-Ppo I and I-Tli I. 
     
     
         32 . The method of  claim 27 , wherein the additional rare restriction enzyme is selected from the group consisting of Acl I, Asc I, AsiS I, BsiW I, Fse I, Mlu I, Not I, Nm I, Pac I, Pme I, Pvu I, Rsr II, Sal I, Sbf I, Sfi I, SgrA I, SnaB I, Swa I, Pl-Sce I, I-Sce I, I-Ceu I, PL-Psp I, I-Ppo I and I-Tli I. 
     
     
         33 . The method of  claim 29 , wherein the additional rare restriction enzyme is selected from the group consisting of Acl I, Asc I, AsiS I, BsiW I, Fse I, Mlu I, Not I, Nm I, Pac I, Pme I, Pvu I, Rsr II, Sal I, Sbf I, Sfi I, SgrA I, SnaB I, Swa I, Pl-Sce I, I-Sce I, I-Ceu I, PL-Psp I, I-Ppo I and I-Tli I.