Restriction digests of pLNbovIL-2 give the following sizes (kb): EcoRI--6.2; SalI--6.2; NcoI--6.2; XbaI--6.0, 0.2; XbaI/SphI--4.3, 1.7, 0.2. Restriction digests of pRK248cIts give the following sizes (kb): EcoRI--11.0; SalI--11.0; NcoI--10.5, 0.5; XbaI--uncut; SphI--11.0. Contains 2 plasmids: pLNbovIL2 (6.2 kb, ampicillin resistant) and pRK248cIts (ATCC 33766) (11.0 kb, tetracycline resistant), encoding the temperature-sensitive lambda cI repressor used to regulate expression from pLNbovIL2. pRK248cIts is present at lower copy number and can usually be distinguished from pLNbovIL-2 on agarose gels stained with ethidium bromide. Detects bovine genomic restriction fragments of the following sizes (kb): BamHI--10.0; EcoRI--5.8, 5.0; HindIII--5.1, 3.3, 1.9. After thermal induction, this construct produces biologically active product. The HgiAI/PstI fragment encoding aa 22 to 135 from pIL-2-4 was blunt-ended and inserted into a construct based on pPL-lambda already containing an oligonucleotide providing a ribosome binding site, an initiator Met codon, and a codon for Ala 21. |
