Pegfp n3 dna

Vectors expressing cell-surface CD28, CD28 fused C-terminally to GFP, cell-surface B7-2 and B7-2 or B7-2C fused C-terminally to Cherry have been described [13 (link), 14 (link)]. Vector expressing B7-1 was generated by cDNA synthesis of human CD80 (NM_005191.3) from total human PBMC RNA using Verso RT-PCR kit (ABgene). CD80 cDNA was generated using KOD polymerase (Novagen) with phosphorylated PCR primers 5′-GACGTCGACATGGGCCACACACGGAGG and 5′-CACGCGGCCGCTTATACAGGGCGTACACTTTCCC. The PCR product was inserted into pEGFP-N3 DNA (Clontech) that had been digested with SalII and NotI and lacked the GFP region, using Fast-Link DNA Ligation Kit (Epicentre). Vector expressing B7-1 fused C-terminally to Cherry was generated from B7-1 cDNA vector template with phosphorylated PCR primers 5′-TACTCGAGATGGGCCACACACGGAGG and 5′-GTCCGCGGTACAGGGCGTACACTTTCCCTTC, deleting the B7-1 termination codon. Upon digestion with XhoI and SacII, the PCR product was inserted into pmCherry-N1 DNA (Clontech).

Vectors expressing cell-surface CD28, CD28 fused C-terminally to GFP, cell-surface B7-2 and B7-2 or B7-2C fused C-terminally to Cherry have been described (4 (link), 5 (link)). Vector expressing B7-1 was generated by cDNA synthesis of human CD80 (NM_005191.3) from total human PBMC RNA using Verso RT-PCR kit (ABgene). CD80 cDNA was generated using KOD polymerase (Novagen) with phosphorylated PCR primers 5′-GAC GTC GAC ATG GGC CAC ACA CGG AGG and 5′-CAC GCG GCC GCT TAT ACA GGG CGT ACA CTT TC CC. The PCR product was inserted into pEGFP-N3 DNA (Clontech) that had been digested with SalII and NotI and lacked the GFP region, using Fast-Link DNA Ligation Kit (Epicenter). Vector expressing B7-1 fused C-terminally to Cherry was generated from B7-1 cDNA vector template with phosphorylated PCR primers 5′-TAC TCG AGA TGG GCC ACA CAC GGA GG and 5′-GTC CGC GGT ACA GGG CGT ACA CTT TCC CT TC, deleting the B7-1 termination codon. Upon digestion with XhoI and SacII, the PCR product was inserted into pmCherry-N1 DNA (Clontech).