Gibson Assembly Calculator:
Enter a DNA sequence into this page and it will calculate the length and GC content, and return the 60 base oligonucleotides you need to perform one variant of the Gibson assembly, developed in the J. Craig Venter Institute. Gibson et al. showed that two or more DNA segments could be rapidly joined together provided there was 20 or more sequence overlap at the end of the sequences to be joined. The original Gibson et al. paper was published in 2009, and showed how DNA segments to be joined were treated with a mixture of a DNA ligase, an 5' exonuclease and a polymerase, a variant of which is now obtainable commercially from New England Biolabs, see here. The exonuclease digests back the 5' region of each overlapping sequence, allowing hybidization of the remaining strands to occur, the polymerase fills sequence gaps and the ligase joins the loose ends, so that in 2 hours or less the desired sequence is generated! A variant of this procedure was used to synthesize the entire mouse mitochondrial genome. To do this a series of 60 base oligonucleotides were generated, the first corresponding to the first 60 bases of the lower or 3' strand of the desired sequence. The next 60 base oligo generates the upper or 5' strand corresponding to bases 40 to 100 of the upper strand, and so overlaps the last 20 bases of the first oligo. The third corresponded to bases 80 to 140 of the lower strand, overlapping the last 20 of the second oligo, and so on down the sequence. Each internal oligonucleotide therefore can hybridize with 20 bases from the previous and 20 bases from the next, and the enzyme mix stiches all these segments together to generate the full length double stranded DNA. In practice Gibson et al. reacted eight 60 base oligonucleotides to produce DNA seqments of total final length ~340 bases. Since each ~340 base segment was designed to overlap in sequence with those flanking it in the genome, these could later be joined together using the same enzyme mix in the same manner. A total of 75 such constructs were fused in several steps to finally produce the entire 16,299 base pair circular mitochondrial DNA sequence. In our simple program sequence should be entered in the single letter DNA code, so A, T, G and C, and it does not matter if the input is upper or lower case or mixed. If you enter a RNA sequence the program will change every U to a T. The program will ignore numbers, spaces, linefeeds and other irrelevant characters, so you can copy sequence directly from GenBank or other DNA sequence databases. The program also takes FASTA format sequence, so will ignore text following the ">" character, the first item in a FASTA file, until there is a line feed/carriage return character, after which it will interpret every A, T, G and C as a base in a DNA sequence. It will then print out a list of the 5' and 3' oligos required to engineer the desired sequence. So all you have to do is paste in your sequence, press to calculate and then copy and paste the output into a wordprocessor or spreadsheet program to email to whoever makes your oligonucleotides. You can also print out the output of the program, but current versions of the JS language used to code this page allow only print out of the entire page. Anyway good luck!