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. 2016 Dec 15;45(7):e52. doi: 10.1093/nar/gkw1261

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© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 1. — Construction of pBACode cloning vectors. (A) A pBACode-1 pool is generated by PCR amplification using pcc2FOS as a template. The 3΄ ends of primers are complementary to the vector sequences flanking the cloning sites so that the whole pcc2FOS vector except for its cloning sites is amplified. The 5΄ ends of the primers carry the cloning sites followed by 20-bp random sequences. (B) A pBACode-2 pool is generated by a combination of PCR amplification and subcloning. First, an intermediate vector is constructed based on pcc2FOS such that its lacZ sequence is replaced by an I-SceI site. The second intermediate vector is constructed by inserting the kan^R selection gene marker into the cloning site of pcc2FOS, and then the lacZ-kan^R-lacZ segment is amplified using primers containing random barcodes. The resulting PCR product is subcloned into the first intermediate vector. Kanamycin selection is used to eliminate no-insert vectors.