e189 Nucleic Acids Research, 2005, Vol. 33, No. 22 PAGE 4 OF 14 Figure 1. Engineering a mini-chromosome with an attB site for the fC31 integrase. (A) The mini-chromosome 49B(A)A9 is 1.8 Mb long and consists mainly of two arrays of repeated sequences. There is a 1.6 Mb array consisting of interspersed DYZ1 and DYZ2 sequences at the end of the mini-chromosome nearest a sub- telomeric puromycin resistance gene. The other end of the mini-chromosome includes sequences necessary for mini-chromosome maintenance in Saccharomyces cerevisiae (ACU), plasmid vector sequences (pBS), a gene conferring resistance to the antibiotic G418 (neo), a 15 kb array of alphoid DNA and a 90 kb array of alphoid DNA. The two alphoid DNA arrays are separated by between 30 and 50 kb of conventional euchromatic DNA that has been cloned and mapped in bacterial vectors. Details of the organization of the mini-chromosome and of the cloning and mapping have been described elsewhere (19). (B) The attB site was targeted into the mini-chromosome with a construct cloned in pBS that included the ACU sequences and homology to the G418 resistance gene. The targeting construct also contained a hygromycin resistance thymidine kinase fusion gene driven by a CCAG promoter with the promoter and coding sequence being interrupted by an attB site for the fC31 integrase. The arrow above the neo in the upper most diagram indicates the position of the SV40 early region promoter driving the G418 resistance gene in this chromosome. Figure 2. Plasmids used to study the activity of the fC31 integrase in DT40 cells. (A) CCAG(50NLS)fC31integrase IRES Zeo is the canonical integrase expression plasmid. It contains the fC31integrase gene either with or without an N-terminal nuclear localization signal cloned into the internal ribosome entry site containing expression vector CCAG IRES Zeo that confers resistance to the antibiotic zeocin. (B) fCattPneo is the promoterless trapping vector in which a coding region potentially capable of conferring resistance to the antibiotic G418 is abutted by a fC31attP site. (C) Apra fC31attPneo/pBAC3e is the BAC vector designed for trapping with the fC31 integrase. As in pBACe3.6 the cloning sites are interrupted by a pUC plasmid.