Table 1.
Parallel Transfer of the Chloramphenicol Acetyl Transferase (CAT) Gene into 12 Destination Vectors
| Function of Destination Vector | Colonies | Control | Correct |
| Expression of native protein in E. coli | 15,000 | 0 | 6/6 |
| His6-fusion protein inE. coli | 10,650 | 0 | 6/6 |
| GST-fusion protein in E. coli | 9200 | 0 | 6/6 |
| Thioredoxin-fusion protein in E. coli | 11,000 | 0 | 6/6 |
| Sequencing, probe synthesis, (+) strand | 13,950 | 0 | 6/6 |
| Sequencing, probe synthesis, (−) strand | 8950 | 0 | 6/6 |
| Transient expression of native protein in mammalian cells | 7950 | 0 | 6/6 |
| Stable expression of native protein in mammalian cells | 10,500 | 1100 | 6/6 |
| Expression of native protein in insect cells | 7800 | 15 | 6/6 |
| Native protein in mammalian cells. Semliki Forest Virus vector | 4150 | 0 | 6/6 |
| His6-fusion protein in insect cells | 6350 | 30 | 6/6 |
| Tetracycline-regulated expression of native protein in mammalian cells | 11,650 | 0 | 6/6 |
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↵Standard vectors were converted to Destination Vectors by inserting a cassette containing the motifsattR1–CmR–ccdB–attR2.
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↵Aliquots (20 or 200 μL) of the 1 ml expression mixture were plated on ampicillin. Colonies are expressed as the number calculated for mL. Cells were competent at 1 × 108/μg pUC.
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↵Identical reactions except the CAT Entry Clone DNA was omitted.
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↵Based on miniprep DNAs (four colonies) and restriction digests (two colonies) from random colonies.
