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B. , D. Strain, and . Dd-strain, pUC vector, DH10B bacterial strains C) ScFv sequences NCBI accession no-(TA10 -AY348549.1) (MB11- AA842745.1) digestion with Not l ? Nhe l. All Fc fragments were ligated in pUX vector at Not l ? Nhe l sites, just downstream to scFv sequence in pUX vector. The ligated product was transformed into competent DH10B strain of E.coli. The positive clone was confirmed by extraction of plasmid DNA of few selected E.coli transformed colonies appeared on selection plate and further, checking the correct insert size of Fc region by digestion with Not l-Nhe l restriction enzymes. Design of scFv+Fc cloning in Dd expression vector and different cloning steps are shown in figure E4. The plasmid DNA of positive clone was transformed in Dd cells (see appendix F) Vector map of pUX-scFv+Fc is shown in figure E5, SF9 -AY348548.1), (F2C-AY348545.1)

E. Figure, isolation of multispecies Fc from phen vector by digesting with Not l -Nhe l restriction enzymes, (c) Screening of clones by restriction digestion using Not l -Nhe l, M ? DNA marker, lane 1-6 (pUX TA10+ Mouse Fc) expected insert 700 bp. Lane 8-11 (pUX TA10+ mCherry Fc) expected insert 1.6kb, (d) Screening of clones by restriction digestion using Not l -Nhe l, pp.1-4