Pbluescript

A 7.9 kb-long random-sequence DNA molecule lacking any nucleosome-positioning sequences nor alpha-satellite DNA sequence was made by taking a fragment of the pBlueScript-1,2,4+pSfv1 plasmid, which consists of fragments of Lambda DNA and a fragment from pSfv1 (Invitrogen) in pBlueScript SK+ (Agilent). Alpha satellite DNA sequence plasmids were obtained as described in [43 (link)]. From this plasmid, DNA fragments each containing 5 alpha satellite sequence repeats were taken and self-ligated to each other to make a final construct of 3.4 kb DNA as a second substrate. For surface immobilization, a 500 bp-long DNA fragment labeled with multiple Dibenzocyclooctyne (DBCO) molecules was ligated at one end of the DNA molecules, while the other end was ligated with a 500 bp-long DNA fragment labeled with multiple biotin molecules for magnetic-bead attachment. In the experiments, we selected, by measuring their torque-responses, rotationally unconstrained DNA molecules that can freely rotate due to the presence of a nick. The complete DNA sequences are given in S1 Text.

Two vectors were designed for targeted integration into the Lmbr1 locus: the first a 7.7 kb homology arm at the 5′ end of the Lmbr1 gene, 7.5 kb upstream of the ZRS; and the second a 7.6 kb arm 74 kb downstream of the ZRS. Mini targeting arms were generated by PCR (the primers are listed in supplementary material Table S1) and cloned using the underlined restriction sites into pBluescript (Invitrogen). Bacterial recombineering (Liu et al., 2003 (link)) was then employed to retrieve the homology arms from the Pac RCPI-21 542n10 (Osoegawa et al., 2000 (link)). These homology arms were subcloned (using the bold restriction sites) into the pLHED vector (Kokubu et al., 2009 (link)). The constructs had been designed with gaps, of 600 and 666 bp, respectively, flanked by HindIII sites that were used to linearise the vectors for targeting.