Takara's DNA cloning vectors pKF, pSTV28, pSTV29, pTV118N, pTWV228 and pUC118 may be used for the following cloning reactions, as described below.
pKF 18k-2/19k-2 DNA
The pKF pUC-derived vector DNA contains two amber stop mutations at the kanamycin resistance gene. When transformed with pKF, supE strains like JM109 but not sup0 strains like MV1184 will grow on kanamycin-containing plates. The pKF DNA may be used for site-directed mutagenesis based on the ODA (Oligonucleotide-directed Dual Amber) method. Using the simplified ODA procedure, a desired mutation can be introduced in only three days. Moreover, pKF DNA is available for target gene cloning and expression via the lac promoter when transformed into supE hosts like JM109. Target genes cloned at the initiation codon (ATG) of the Nde I restriction site (CATATG) have a higher translation efficiency.
pSTV 28/29 DNA
The pSTV vectors contain a beta-galactosidase gene, a pACYC184 origin of replication, a chloramphenicol resistance gene and the pUC119 multiple cloning site. Because the pSTV plasmids produce fewer copy numbers compared with pUC-type high copy number plasmids, they are suitable for the expression of genes that may be toxic to host cells. The pSTV 28/29 vectors contain the pACYC184 origin of replication and can be co-transformed with pUC or pBR vectors.
pTV118N DNA is a phagemid vector constructed from a modified pUC118. This plasmid contains the sequence CCATGG which includes the cleavage sequence for the restriction enzyme NcoI as well as the start codon (ATG) for lacZ. This enables target gene expression at the NcoI site. Protein expression is enabled by the vector's lac promoter. The pTV118N vector also contains a lacZ SD sequence. There are eight bases between the lacZ SD sequence and the initiation codon, allowing high level expression of target genes. Induction of single-stranded DNA by helper phage and its subsequent sequencing with RV-N primer enables accurate sequencing from the start codon site, ensuring an insert's correct translation frame.
The pTWV228 vector contains a pBR322 origin of replication, an ampicillin-resistance gene, an intergenic region (IG region) of the M13 phage DNA and a beta-galactosidase gene containing the multiple cloning site of pUC118. The vector is low-copy number, which makes it useful during the expression of genes that present potential toxicity to their host.
pUC118 is a plasmid vector intended for the preparation of single-stranded DNA. pUC118 DNA was constructed by inserting the intergenic region (IG region) of the M13 phage DNA into a pUC18 plasmid. Co-transformation of pUC118 with the helper phage M13K07 induces the production of single-stranded DNA that is packaged into phage particles and released from bacterial cells. There is almost no contamination by the single-stranded helper phage DNA. Using this system, large (up to 7 kb) and deletion-free single-stranded DNA can be stably obtained.