PAGE 3 OF 9 plasmids are named pTnLoxP511(B)RSVneo 1 or pTnLoxP511(B)RSVneo 2 to distinguish the two orientations of the RSVneo cassette. Only pTnLoxP511(B)RSVneo 2 is discussed here. Construction of a Tn10 minitransposon with wild-type loxP sites The kanamycin resistance gene-containing wild-type loxP transposon plasmid, pTn(RSVneo 2)/loxP, was constructed by introducing a RSV neomycin gene cassette into the previ- ously described pTnMarkerless2 transposon plasmid (28). It differs from pTnBAC/loxP (14) in that all sequences related to the chloramphenicol resistance gene were removed from the latter, in order to reduce recombination between the chloram- phenicol resistance gene in BACs and those sequences in the pTnBAC/loxP plasmid (14) [see discussion (28)]. The RSV neomycin gene cassette was excised from pTnBAC/loxP with AscI, the DNA fragment puriﬁed from an agarose gel, and ligated to pTnMarkerless2 linearized also with AscI. Plasmids containing both orientations of the RSV neo gene cassette were isolated, pTn(RSVneo 1)/loxP and pTn(RSVneo 2)/loxP, and only the latter orientation used here and shown in Figure 1. Generating deletions from the loxP511 end of inserts in pBACe3.6 vector-derived clones The following 10 clones from the human and mouse BAC libraries were obtained from BACPAC resources, CHORI (Oakland, CA): RP11-219A15, RP11-158M20, Nucleic Acids Research, 2005, Vol. 33, No. 13 e118 RP11-434D2, RP23-209O22, RP23-92L23, RP23-366M16, RP23-101N20, RP23-124B2, RP23-444K15, RP23-130D16. The genomic inserts in these clones are ﬂanked by a wild- type and a mutant loxP511 site in the BAC vector pBACe3.6 (9–11,22). Nested deletions using pTnLoxP511(B)markerless 1, pTnLoxP511(B)markerless 2 and pTnLoxP511(B)RSVneo 2 from the loxP511 end were generated in each of the above clones as previously described (28,29). Processing multiple BAC clones to make end-deletions BACs were also pooled together and processed together in a single tube to make end-deletions. Each BAC clone was transformed separately with the transposon plasmid, and the transformed colonies grown to saturation before pooling. End-sequencing of BAC deletion clones Typically, miniprep DNA was isolated from 60 clones picked randomly from the several hundred member BAC deletion library. Deletion clones were passed through an ampicillin sensitivity screen (20) if the transposon plasmid was recovered in more than 20% of the deletions. Approximately half the clones from the ﬁrst round deletion series analyzed on FIGE were of unique size. Deletions arising from intra-insert recombinations independent of loxP-Cre, seen only when using markerless transposons of either type, were weeded out after FIGE analysis (lane 1 of Figure 2 shows an e.g.). These are identiﬁed by their BAC vector DNA fragment Deleting From the loxP511 end of DNA insert in pBACe3.6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 80- 70- 60- 50- 40- 30- 20- 10- 5- Figure 2. FIGE analysis of NotI digested DNA isolated from progressive truncations made from one or both ends of insert DNA. Left panel: DNA isolated from deletion clones generated with either pTnLoxP511(B)markerless 1 (lanes 3–15) or pTnLoxP511(B)RSVneo 2 (lanes 19–30) in BAC clone RP23-444K15 and RP11- 219A15, respectively, were analyzed by FIGE. Lane 17 contains the DNA from starting BAC clone RP23-444K15, while lanes 2, 18 and 41 contain 5 kb DNA ladders. Right panel: Second round deletions made with pTn(RSVneo 2)/loxP from the wild-type loxP end of insert DNA from the deletion clone shown in lanes 3 and 31. Lanes 32–40 show deletions, while 42–48 show inversions. Deletions with pTnLoxP511(B)RSVneo 2 (lanes 19–30) produce a 10.5 kb BAC vector DNA fragment, while those with pTnLoxP511(B)markerless 1 (lanes 3–15) produce no BAC vector band because one of the NotI sites is lost as a result of the deletion (Figure 3). Intra-insert deletions independent of LoxP-Cre recombination produce the same sized BAC vector band as starting BAC (lane 1).