Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning
Phage lambda cDNA cloning vectors for subtractive hybridization, fusion-protein synthesis and Cre-loxP automatic plasmid subcloning
- Gene, 88(1), p.25-36, 1990 .
We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the EXLX(+)and EXLX(-)vectors that can be used for the expression in Escherichia coli of protein encoded by cDNA inserts is achived by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage arms, LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of specialized plasmid between these items sites will convert it into a phage cDNA cloning vector with automatic plasmid subcloning capability.
RECOMBINANT DNA
BACTERIOPHAGES
T7
F1
P1
E. COLI RNA POLYMERASE
PROMOTERS
SITE-SPECIFIC RECOMBINATION
We describe the construction and use of two classes of cDNA cloning vectors. The first class comprises the EXLX(+)and EXLX(-)vectors that can be used for the expression in Escherichia coli of protein encoded by cDNA inserts is achived by the fusion of cDNA open reading frames to the T7 gene 10 promoter and protein-coding sequences. The second class, the SHLX vectors, allows the generation of large amounts of single-stranded DNA or synthetic cRNA that can be used in subtractive hybridization procedures. Both classes of vectors are designed to allow directional cDNA cloning with non-enzymatic protection of internal restriction sites. In addition, they are designed to facilitate conversion from phage to plasmid clones using a genetic method based on the bacteriophage P1 site-specific recombination system; we refer to this as automatic Cre-loxP plasmid subcloning. The phage arms, LOX, used in the construction of these vectors have unique restriction sites positioned between the two loxP sites. Insertion of specialized plasmid between these items sites will convert it into a phage cDNA cloning vector with automatic plasmid subcloning capability.
RECOMBINANT DNA
BACTERIOPHAGES
T7
F1
P1
E. COLI RNA POLYMERASE
PROMOTERS
SITE-SPECIFIC RECOMBINATION