Dna cloning vector plasmids and methods for their use

Abstract

The present invention is a group of cloning vector plasmids for use in constructing DNA molecules, such as transgenes, for the purpose of gene expression or analysis of gene expression. The present invention is also a method for using the cloning vector plasmids in a variable series of cloning steps to produce a final transgene product. 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 . (canceled) 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . (canceled) 
     
     
         7 . (canceled) 
     
     
         8 . A shuttle vector containing a multiple cloning site (MCS) for cloning in DNA fragments, comprising
 a) a first set of at least two unique rare restriction sites that define the 5′ portion of the variable MCS;   b) a variable MCS comprising any set of restriction sites that are unique to the shuttle vector said first set of unique rare restriction sites being 5′ to the variable MCS; and   c) a second set of at least two unique rare restriction sites that define the 3′ portion of the variable MCS,   
       wherein each rare restriction site differs from any other. 
     
     
         9 . The shuttle vector according to  claim 8 , wherein said unique rare restriction sites of said first and second sets of unique rare restriction sites are selected from the group consisting of AsiSI, SgrAI, PacI, AscI, MluI, SnaBI, NotI, SalI, SwaI, RsrII and BsiWI. 
     
     
         10 . The shuttle vector according to  claim 9 , further comprising at least ten unique common restriction sites selected from the group consisting of AatII, AflIII, ApaI, AvrII, BlpI, BamHI, BspEI, BspHI, Eco0109I, EcoRV, EcoRI, HindIII, KpnI, NheI, NgoMIV, PmeI, PvuI PvuII, PspOMI, SapI, SphI, XbaI, and XhoI. 
     
     
         11 . A vector containing at least one module of DNA fragments, said vector comprising
 a) a first homing endonuclease site;   b) a first set of at least two unique rare restriction sites, said first homing endonuclease site located 5′ to said at least two unique rare restriction sites;   c) a DNA fragment module, said first set of at least two unique rare restriction sites located 5′ to said module;   d) a second set of at least two unique rare restriction sites said module located 5′ to said at least two unique rare restriction sites; and   e) a second homing endonuclease site, said second set of at least two unique rare restriction sites located 5′ of said second homing endonuclease site,   
       wherein said first and second sets of unique rare restriction sites are different from one another. 
     
     
         12 . The vector according to  claim 11 , wherein each rare restriction site is selected from the group consisting of AsiSI, SgrAI, PacI, AscI, MluI, SnaBI, NotI, SalI, SwaI, RsrII, and BsiWI. 
     
     
         13 . The vector according to  claim 12 , wherein said DNA fragment module is selected from the group of naturally occurring or synthetic sequences consisting of a primer site sequence, a chromatin modification domain sequence, a 3′ regulatory sequence, a promoter sequence. protein-coding sequence, reporter gene, an enhancer sequence, an intron, an exon, a poly-A tail sequence, a multiple cloning site sequence, a localization signal, and an mRNA stabilization signal sequence. 
     
     
         14 . A plasmid comprising a polynucleotide sequence, wherein said sequence comprises
 a. a first set of restriction sites, said first set comprising at least two common restriction sites that are unique within said plasmid;   b. a second set of restriction sites, said second set comprising at least two rare restriction sites that are unique within said plasmid;   c. a first region of acceptor nucleotide sequence;   d. a third set of restriction sites, said third set comprising at least two rare restriction sites that are unique within said plasmid; said second and third sets of restriction sites being separated by said first region of acceptor nucleotide sequence;   e. a second region of acceptor nucleotide sequence;   f. a fourth set of restriction sites, said fourth set comprising at least two rare restriction sites that are unique within said plasmid; said third and fourth sets of restriction sites being separated by said second region of acceptor nucleotide sequence;   g. a third region of acceptor nucleotide sequence;   h. a fifth set of restriction sites, said fifth set comprising at least two rare restriction sites that are unique within said plasmid, said fourth and fifth sets of restriction sites being separated by said third region of acceptor nucleotide sequence; and   i. a sixth set of restriction sites, said sixth set comprising at least two common restriction sites that are unique within said plasmid,   
       wherein elements (a) through (i) are arranged sequentially in the 5′ to 3′ direction of said plasmid. 
     
     
         15 . The plasmid of  claim 14 , wherein said plasmid further comprises at least one 5′ primer site and at least one 3′ primer site. 
     
     
         16 . The plasmid of  claim 15 , wherein said plasmid further comprises a first homing endonuclease site that is 5′ to said first set of restriction sites. 
     
     
         17 . The plasmid of  claim 16 , wherein said plasmid further comprises a second homing endonuclease site that is 3′ to said sixth set of restriction sites. 
     
     
         18 . The plasmid of  claim 17 , wherein said second homing endonuclease is in opposite orientation to said first homing endonuclease site. 
     
     
         19 . The plasmid of  claim 18 , wherein said first and second homing endonuclease sites are cleavable by the same homing endonuclease enzyme. 
     
     
         20 . The plasmid of  claim 17 , wherein said first and second homing endonuclease sites are cleavable by two different homing endonuclease enzymes. 
     
     
         21 . The plasmid of  claim 17 , wherein said first set of restriction sites comprises at least three common restriction sites. 
     
     
         22 . The plasmid of  claim 17 , wherein said sixth set of restriction sites comprises at least three common restriction sites. 
     
     
         23 . The plasmid of  claim 22 , wherein said second set of restriction sites comprises at least three rare restriction sites. 
     
     
         24 . The plasmid of  claim 23 , wherein said third set of restriction sites comprises at least three rare restriction sites. 
     
     
         25 . The plasmid of  claim 24 , wherein said fourth set of restriction sites comprises at least three rare restriction sites. 
     
     
         26 . The plasmid of  claim 25 , wherein said fifth set of restriction sites comprises at least three rare restriction sites. 
     
     
         27 . The plasmid of  claim 14 , wherein said common restriction sites of said first and sixth sets of restriction sites are polynucleotide sequences that are cut by the restriction enzymes selected from the group consisting of AatII, AflIII, ApaI, AvrII, BlpI, BamHI, BspEI, BspHI, Eco0109I, EcoRV, EcoRI, HindIII, KpnI, NheI, NgoMIV, PmeI, PvuI PvuII, PspOMI, SapI, SphI, XbaI, and XhoI. 
     
     
         28 . The plasmid of  claim 14 , wherein said rare restriction sites of said second, third, fourth and fifth sets of restriction sites are polynucleotide sequences that are cut by the restriction enzymes selected from the group consisting of AsiSI, SgrAI, PacI, AscI, MluI, SnaBI, NotI, SalI, SwaI, RsrII and BsiWI. 
     
     
         29 . The plasmid of  claim 17 , wherein said first and said second homing endonuclease sites are sites that are cleavable by the homing endonucleases selected from the group consisting of I-SceI, PI-SceI, I-CeuI and PI-PspI. 
     
     
         30 . A plasmid comprising a polynucleotide sequence, wherein said sequence comprises
 a. a first BstX I restriction site;   b. a first region of acceptor nucleotide sequence;   c. a second BstX I restriction site, said second BstX I restriction site being in opposite orientation to said first BstX I restriction site;   d. a first homing endonuclease site, wherein said first homing endonuclease site is not cleavable by BstX I;   e. a second region of acceptor nucleotide sequence;   f. a second homing endonuclease site that is in opposite orientation to said first homing endonuclease site, wherein said second homing endonuclease site is the same as said first homing endonuclease site; and   
       wherein elements (a) through (f) are arranged sequentially in the 5′ to 3′ direction of the plasmid. 
     
     
         31 . The plasmid of  claim 30 , wherein the plasmid further comprises a first 5′ primer site and a first 3′ primer site. 
     
     
         32 . The plasmid of  claim 30 , wherein said first 5′ primer site is 5′ to said BstX I site and said first 3′ primer site is 3′ to said second homing endonuclease site. 
     
     
         33 . The plasmid of  claim 31 , wherein the plasmid further comprises at least a first chromatin modification domain. 
     
     
         34 . The plasmid of  claim 31 , wherein the plasmid comprises at least a first and second chromatin modification domain, wherein said first chromatin modification domain is 5′ to said first BstX I site and said second chromatin modification domain is 3′ to said second homing endonuclease site. 
     
     
         35 . A plasmid capable of receiving at least two transgenes, the plasmid comprising a polynucleotide sequence, wherein said polynucleotide sequence comprises
 a. a first homing endonuclease site;   b. a region of acceptor nucleotide sequence,   
       wherein said region of acceptor nucleotide sequence is flanked by sites recognized by BstX I; and
 c. a second homing endonuclease site, 
 
       wherein elements (a) through (c) are arranged in 5′ to 3′ order within the plasmid. 
     
     
         36 . The plasmid of  claim 35 , wherein said BstX I sites are in opposite orientation. 
     
     
         37 . The plasmid of  claim 35 , further comprising at least one polynucleotide sequence selected from the group consisting of a primer site sequence, a chromatin modification domain sequence, a recombination arms sequence, and a selector gene sequence. 
     
     
         38 . A vector expression system comprising a shuttle vector of  claim 8  and a vector of  claim 10 . 
     
     
         39 . A kit for cloning a gene of interest, said kit comprising a shuttle vector, a vector, and instructions.