The pGEM?-11Zf(?) Vector(a) can be used as a standard cloning vector, as a template
for in vitro transcription and for the production of ssDNA. The plasmid contains T7 and
SP6 RNA polymerase promoters flanking a multiple cloning region within the α-peptide
coding region of β-galactosidase (1). Insertional inactivation of the α-peptide allows
recombinant clones to be directly identified by color screening on indicator plates when
using appropriate E. coli strains (e.g., JM109, DH5α?, XL1-Blue). The multiple cloning
region contains unique restriction sites for Sfi I, Sac I, EcoR I, Sal I, Xho I, BamH I,
Apa I, Xba I, Not I, Sph I, Nsi I and Hind III.
For induction of ssDNA, bacterial cells with the F′ episome (e.g., JM109, XL1-Blue,
DH5α?F′) containing pGEM?-11Zf(?) recombinants are infected with an appropriate
helper phage. The plasmid then enters the f1 replication mode, and the resulting
ssDNA is exported from the cell as an encapsidated virus-like particle. The sequence of
the ssDNA rescued upon infection with helper phage is the same strand as that shown
in Figure 1. The exported ssDNA can be used for in vitro mutagenesis or can be
sequenced using Promega's SP6 Promoter Primer or pUC/M13 Reverse Primer.
The sequences of Promega vectors are available online at www.promega.com/vectors/
and are also available from the GenBank? database.