Figure 1
From: Directed PCR-free engineering of highly repetitive DNA sequences
Cloning strategy of Poly-Q encoding sequences. (A) Annealed oligonucleotides used for cloning of the Poly-Q encoding regions. The recognition sequences and the corresponding cleavage sites of the restriction endonucleases are indicated by different colors. The orientation of the two inward facing Type IIS restriction sites (BsaI and BsmBI) results in cleavage within the Poly-Q encoding sequence and allows seamless elongation of the Poly-Q stretch. (B) Detailed representation of the elongation cycles. Digestion of the double-stranded oligonucleotides (BsaI/SacI) and the vector (BsmBI/SacI) resulted in compatible overhangs allowing for seamless elongation. Used restriction sites are underlined. (C) The annealed oligonucleotides were initially subcloned using BsaI and SacI and subsequently inserted into pMK1 via NdeI and SacI. For further elongation BsaI/SacI digested oligonucleotides were ligated into pMK1-Qn digested with BsmBI and SacI. Thereby the number of Q-encoding nucleotides was increased by nine and a new BsmBI site was introduced allowing subsequent rounds of elongation.