Methods and compositions for seamless cloning of nucleic acid molecules

A vector comprising:

• (a) one or more toxic genes;
• (b) one or more type IIs restriction enzyme recognition sites;
• and
• (c) one or more site-specific recombination sites.

A method of regulating the expression of one or more genes in a transgenic cell or a transgenic animal using interfering RNA, comprising:

• (a) identifying one or more target nucleic acid sequences in said cell or animal;
• (b) preparing one or more nucleic acid molecules which encode one or more interfering RNAs, wherein said interfering RNAs bind to said one or more target nucleic acid sequences;
• (c) combining
• (i) one or more first nucleic acid molecules encoding one or more interfering RNAs flanked by one or more first type IIs restriction enzyme recognition sites;
• (ii) one or more second nucleic acid molecules comprising one or more selectable markers flanked by one or more second type IIs restriction enzyme recognition sites;
• and
• (iii) one or more site-specific type IIs restriction enzymes;
• and
• (d) incubating said combination under conditions sufficient to join one or more of said one or more nucleic acid molecules encoding one or more interfering RNAs and one or more of said second nucleic acid molecules, thereby producing one or more desired product nucleic acid molecules;
• (e) inserting said one or more interfering RNA-containing product nucleic acid molecules into said cell or one or more cells of said animal, under conditions such that said one or more interfering RNAs bind to said one or more target nucleic acid sequences, thereby regulating expression of said one or more genes.