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Vector for constructing cDNA libraries

Vector permitting positive selection for inserts

Vector permitting production of single-stranded DNA

Restriction digests of the clone give the following sizes (kb): HindIII--24.1, 17.6; EcoRI--22.0, 20.0; BamHI--24.8, 16.9; XbaI--32.7, 9.0; NotI--41.7.

Efficiency of phagemid recovery is approximately 20%. Plasmid pCRE1 may be a low level contaminant, but is easily distinguished from pMGU DNA.

To prepare phagemid from lambdaMGU2, grow recombinants on a RecA- host expressing the Cre protein (E. coli 1046[pCRE1], ATCC 77368) and select for ampicillin resistance. The pMGU product is 4.185 kb.

Vector useful for constructing cDNA libraries. Permits positive selection for inserts using the Spi- phenotype, and excision of phagemid by lox/cre site-specific recombination.

To enable the positive selection of inserts, the library should be plated on a P2 lysogen such as Escherichia coli Q359 (ATCC 47019).

Inserts can be amplified using the following primers flanking the BamHI cloning site: upstream 5'-AAGAGGCAGAACTGGCAG-3' and downstream 5'-ATCGATGCATAGCGATTC-3'.

The order of the major features in the cloning region of the lambda vector is: lambda J - SmaI - SalI - loxP - EcoRI - M13 ori - ampR - pMB1 ori - HindIII - 3'gam/BamHI/5'gam - XhoI - loxP - SalI - lambda N.