Cloning vectors

2. Activation domain vector: pOAD (with uniques restriction sites and main features)

Sequence features:

• LEU2: 3831-2740
• beta-lactamase (amp resistance): 4459-5313
• CEN4: 652-1505
• ARS1 (?): 1534-1750 (corresponds to sequence between TRP1 and GAL3 on yeast chromosome 4)
• GAL4 (AD): 7782-8215 (first 66 bp not from Gal4)

Cloning site (position 8144-56):

C TAT CTA TTC GAT GAT GAA GAT ACC CCA CCA AAC CCA AAA AAA GAG ATC GAA TTC CAG CTG ACC ACC ATG GCA ATT CCC GGG GAT CCG TCG ACC TGC AGA GAT CTA TGA ATC GTA GAT ACT GAA AAA C[CC CGC AAG]

This sequence corresponds to the two re-PCR primers (green + blue = forward primer, pink + black = reverse primer) except for the very 5' 8 bp of the reverse primer which are shown in brackets. The two restriction sites for linearizing pOBD2 (PvuII = CAGCTG and NcoI = CCATGG) are underlined. In the 4 bp-region of mixed color (blue and pink) the two primer sequences overlap). The primers and vectors are designed in a way that a single re-PCR product can be used for both the bait (pOBD2) and prey (pOAD) clones.

The green and blue sequences here correspond to the red and grey sequences in the figure below. Pink and black correspond to beige and grey (sorry for the confusing color codes).

Actual sequence of pOAD.

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