Table 1.

Primer sets used in PCR reactions for generation of SP-CC120/121G insert construct

SP-C insert name (amino acid) PCR primers
5′ primer (forward)*3′ primer (reverse)
Primary PCR
Met1-Lys125(A) dTCCGGACTCAGATCTATGGATGTGGGCAGCAAAGAGGTCCTG(B) dCTTCATGATGTAGCCGCCGGTGCCAGGGGC
Ala116-Ile197(C) dGCCCCTGGCACCGGCGGCTACATCATGAAG(D) dCTATAGGGATCCGCCCTCTAGATGCATGCTCGAGCG
Secondary PCR
SP-CC120/1212G(A) dTCCGGACTCAGATCTATGGATGTGGGCAGCAAAGAGGTCCTG(D) dCTATAGGGATCCGCCCTCTAGATGCATGCTCGAGCG
  • For both reactions of primary PCR, pcDNA3-hSP-C(6+) was used as template.

    Completely overlapping complementary primers B and C. which permit complementation in the secondary PCR, are highlighted in bold.

  • * Primer A contains a BglII site (5′-AGATCT-3′) for in-frame ligation into EGFP-C1.

  • The 3′ end of primer D matches the polylinker of pcDNA3 and contains a BamHI site (5′-GGATCC-3); use of this primer resulted in inclusion of the 3′ untranslated region in the mutant construct.