Figure S1: Maps of the intermediate plasmids used for cloning double TBR binary vector SKU211.

Figure S2: FLP-mediated excision of TBRs from a double TBR construct

(A) Schematic representation of double TBR construct with tobacco Rb7 TBR inserted both on 5´ and 3´ of the 35S-Luciferase:GFP fusion construct. Each TBR is flanked by a pair of directly oriented FRT sites (small red arrow). Note that the FRT sites in each pair are in the direct repeat orientation to permit excision of the intervening TBR, but the pairs are inverted so that the two individual FRT sites remaining after both TBRs are excised will be in inverted orientation. Black arrows indicate the primers used for the PCR analyses in panels C, D and E. Expected products are indicated by the thin black lines, with sizes indicated in base pairs (bp).

(B) Products expected from FLP action on the double TBR construct. In addition to excising the two TBRs, FLP will reversibly invert the Luciferase-GFP transgene without excising it, creating a mixture of the two arrangements shown in the figure. Black arrows indicate primer sites and lines indicate expected products, as in Panel A. Note that primers 1 and 2 are only expected to produce a product when Luciferase-GFP transgene is inverted.

(C, D & E) PCR analysis of DNA from FLP-expressing or wild-type E. coli, showing excision of both 5’ and 3’ TBRs, together with inversion of the LUC:GFP reporter between remaining FRT sites. Either wt (lanes 4 and 8) or FLP-expressing E. coli (lanes 1-3 and 5-7) were transformed with double TBR binary vector shown in panel A. The same DNA samples were used for panels C, D, and E. Panel C shows that primers 1 and 3 amplify either a 2700 bp fragment prior to 3’TBR excision, or a 1533 bp fragment after 3’TBR excision, as expected. Panel D shows that primers 1 and 2 amplify a 400 bp fragment only when the construct is inserted in FLP-expressing E. coli. Amplification of this fragment depends on inversion of the LUC:GFP region between the internal FRT sites; it does not occur in wild type E. coli. Panel E shows that primers 2 and 4 amplify either a 2900 bp fragment prior to 5’TBR excision (Lanes 4 & 8), or a 1700 bp fragment after 5’TBR excision (Lanes 1-3 & 5-7). The PCR products described here can be obtained from the same DNA only when both TBRs are excised and the LUC:GFP fragment is present in both orientations.

Table S1:List of Primers Used for Luc-GFP Fusion
Primer Name / Sequence
HindIII-attR1-F / 5' - ACT GTC GAC AAG CTT ACA AGT TTG TAC AAA AAA GCT G - 3'
Nsi-attR2-R / 5' - CCA ATG CAT ACC ACT TTG TAC AAG - 3'
12attB1-5'35SPfor / 5'-AAA AAG CAG GCT AGA TTA GCC TTT TCA ATT TCA G - 3'
attB1 / 5' - ACA AGT TTG TAC AAA AAA GCA GGC T- 3'
12attB2-3'NOSTrev / 5' -AGA AAG CTG GGT GAT CTA GTA ACA TAG ATG ACA CCG - 3'
attB2 / 5' - ACC ACT TTG TAC AAG AAA GCT GGG T- 3'
3'Luc-5'GFP-F / 5' -AAG GGC GGA AAG TCC AAA TTG ATG AGT AAA GGA GAA GAA CTT - 3'
5'GFP-3'Luc-R / 5' -AAG TTC TTC TCC TTT ACT CAT CAA TTT GGA CTT TCC GCC CTT - 3'
Table S2:List of Primers Used for Q-PCR Copy Number Estimation
Primer/Probe Name / Sequence
LUC-F / 5'- CGC GTT ATT TAT CGG AGT TG - 3'
LUC-R / 5'- CTG CGA AAT GTT CAT ACT GT - 3'
LUC-Probe / 5'-FAM-TTG CGC CCG CGA ACG ACA TTT AT-3BHQ_1- 3'
Genta-F / 5'- TGC TCC GTA GTA AGA CA - 3'
Genta-R / 5'- ACC TGG GCA GAA CGT AAG - 3'
Genta-Probe / 5'-FAM-TTC ATC GCG TTG CTG CCT TC-BHQ_1- 3'
4HPPD -F / 5'- GAG CAG TAT TGG AGG ATT C - 3'
4HPPD -R / 5'- CGA CCC GTT TCT TGA G - 3'
4HPPD -Probe / 5'-Hex-CTT CAT GCC TTC TCC TCC GCC TA-BHQ_1- 3'