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III and Smith HO (2008) Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319:1215–1220. 7. Au LC, Yang FY, Yang WJ, Lo SH and Kao,CF (1998) Gene synthesis by a LCR-based approach: High-level production of leptin-L54 using synthetic gene in Escherichia coli. Commun 248:200–203. 8. Stemmer WP, Crameri A, Ha KD, Brennan TM and Heyneker HL (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53.

Each node in the tree represents a biochemical process with a product and two precursors. The algorithm starts with the root of the tree (target molecule) and for each node checks whether its product sequence exists with no errors in one of the clones. If such a clone exists, this product is marked as a new basic building block for reconstruction of the target molecule, and its primer pair and relevant clone (as template) are registered as its generating PCR reaction. If there is no clone which contains an error-free sequence of the node product, the reaction is registered as existing reaction in the new protocol, and the algorithm is recursively executed on the two precursors of the product.

7. Sequencing of the true smPCR clones. 8. Computation of minimal cut from clones and reconstruction of the target molecule from the error-free segments, as shown in the manuscript and more generally in ref. 5. 2. Cloning Fragments are cloned into the pGEM-T Easy Vector System 1. Vectors containing cloned fragments are transformed into JM109 competent cells and sequenced. 3. Single-Molecule PCR smPCR is performed with hot-start Accusure for the longer mitochondrial DNA and with Taq polymerase for the GFP fragment.

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