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. 2017 Oct 27;7:14261. doi: 10.1038/s41598-017-14496-5

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Assembly of a 7.09 kb DNA molecule by generating the blunt end of the assembly entrance. The stitching sites GGG are boxed. The black line and lowercase sequence represents pLDR. The red line and uppercase sequence represents the first fragment of PF1, and orange represents the second fragment of PF2. (a) The 7.09 kb DNA molecule was divided into two fragments by 1 stitching site GGG and the matching overlapping primers. (b) The first round of enzymatic assembly reactions for inserting PF1 is shown. The backbone vector of pLDR was digested by SmaI. The SmaI site was then eliminated in the forward primer of P1-R′ (P1-R′ was a reverse complement with P1-R), and a SmaI site (cccGGG) was restored by splicing the first stitching site and overlapping region in the reverse primer of P1-F. When the first fragment of PF1 obtained by PCR with primers of P1-F and P1-R was assembled into pLDR, there was only 1 SmaI site in the first round of the assembled products, which could be cut to generate the assembly entrance for the second round. (c) The second round of the enzymatic assembly reaction for inserting PF2 is shown. The intermediate plasmid of pLDR-PF1 was digested by SmaI. As for the first stitching site GGG in P2-R′ (P2-R′ was a reverse complement with P2-R), the second fragment of PF2 must join together with the first fragment of PF1 seamlessly.