LtrB TargeTron Design December 01 2012 Predicted insertion sites for lacZ: -1|+1 -30 -1 LtrB +1 +15 score 635|636s TATGTGGCGGATGAGCGGCATTTTCCGTGA gtgc...tcac CGTCTCGTTGCTGCA 8.7 1063|1064a CATGCAGAGGATGATGCTCGTGACGGTTAA gtgc...tcac CGCCTCGAATCAGCA 9.1 1125|1126s CAGGTCATGGATGAGCAGACGATGGTGCAG gtgc...tcac GATATCCTGCTGATG 9.1 1542|1543s GCGCGCGTGGATGAAGACCAGCCCTTCCCG gtgc...tcac GCTGTGCCGAAATGG 9.2 2329|2330a GGGTGAACTGATCGCGCAGCGGCGTCAGCA gtgc...tcac GTTGTTTTTTATCGC 8.2 lacZ length: 3075 b.p. Target sites are indicated by nucleotide residues bracketing the intron-insertion site, with "s" and "a" denoting the sense-strand and antisense-strand, respectively. PCR primers to modify the TargeTron for insertion into predicted sites: 635|636s IBS1/2 AAAAAAGCTTATAATTATCCTTACATTTCCCGTGAGTGCGCCCAGATAGGGTG 635|636s EBS1/delta CAGATTGTACAAATGTGGTGATAACAGATAAGTCCCGTGACGTAACTTACCTTTCTTTGT 635|636s EBS2 TGAACGCAAGTTTCTAATTTCGATTAAATGTCGATAGAGGAAAGTGTCT 1063|1064a IBS1/2 AAAAAAGCTTATAATTATCCTTACGTGACGGTTAAGTGCGCCCAGATAGGGTG 1063|1064a EBS1/delta CAGATTGTACAAATGTGGTGATAACAGATAAGTCGGTTAACGTAACTTACCTTTCTTTGT 1063|1064a EBS2 TGAACGCAAGTTTCTAATTTCGATTTCACGTCGATAGAGGAAAGTGTCT 1125|1126s IBS1/2 AAAAAAGCTTATAATTATCCTTAACGATCGTGCAGGTGCGCCCAGATAGGGTG 1125|1126s EBS1/delta CAGATTGTACAAATGTGGTGATAACAGATAAGTCGTGCAGGATAACTTACCTTTCTTTGT 1125|1126s EBS2 TGAACGCAAGTTTCTAATTTCGATTATCGTTCGATAGAGGAAAGTGTCT 1542|1543s IBS1/2 AAAAAAGCTTATAATTATCCTTACAGCCCTTCCCGGTGCGCCCAGATAGGGTG 1542|1543s EBS1/delta CAGATTGTACAAATGTGGTGATAACAGATAAGTCTTCCCGGCTAACTTACCTTTCTTTGT 1542|1543s EBS2 TGAACGCAAGTTTCTAATTTCGATTGGCTGTCGATAGAGGAAAGTGTCT 2329|2330a IBS1/2 AAAAAAGCTTATAATTATCCTTAGCGGCCTCAGCAGTGCGCCCAGATAGGGTG 2329|2330a EBS1/delta CAGATTGTACAAATGTGGTGATAACAGATAAGTCTCAGCAGTTAACTTACCTTTCTTTGT 2329|2330a EBS2 TGAACGCAAGTTTCTAATTTCGATTGCCGCTCGATAGAGGAAAGTGTCT EBS Universal primer AATTAGAAACTTGCGTTCAGTAAACACAACTTATAC gBlocks Gene Fragments to modify the TargeTron for insertion into predicted sites: 635|636s gBlock TTCCCCTCTAGAAAAAAGCTTATAATTATCCTTACATTTCCCGTGAGTGCGCCCAGATAGGGTGTTAAGTCAAGTAGTTTAAGGTACTACTCTGTAAGATAACACAGAAAACAGCCAACCTAACCGAAAAGCGAAAGCTGATACGGGAACAGAGCACGGTTGGAAAGCGATGAGTTACCTAAAGACAATCGGGTACGACTGAGTCGCAATGTTAATCAGATATAAGGTATAAGTTGTGTTTACTGAACGCAAGTTTCTAATTTCGATTAAATGTCGATAGAGGAAAGTGTCTGAAACCTCTAGTACAAAGAAAGGTAAGTTACGTCACGGGACTTATCTGTTATCACCACATTTGTACAATCTG 1063|1064a gBlock TTCCCCTCTAGAAAAAAGCTTATAATTATCCTTACGTGACGGTTAAGTGCGCCCAGATAGGGTGTTAAGTCAAGTAGTTTAAGGTACTACTCTGTAAGATAACACAGAAAACAGCCAACCTAACCGAAAAGCGAAAGCTGATACGGGAACAGAGCACGGTTGGAAAGCGATGAGTTACCTAAAGACAATCGGGTACGACTGAGTCGCAATGTTAATCAGATATAAGGTATAAGTTGTGTTTACTGAACGCAAGTTTCTAATTTCGATTTCACGTCGATAGAGGAAAGTGTCTGAAACCTCTAGTACAAAGAAAGGTAAGTTACGTTAACCGACTTATCTGTTATCACCACATTTGTACAATCTG 1125|1126s gBlock TTCCCCTCTAGAAAAAAGCTTATAATTATCCTTAACGATCGTGCAGGTGCGCCCAGATAGGGTGTTAAGTCAAGTAGTTTAAGGTACTACTCTGTAAGATAACACAGAAAACAGCCAACCTAACCGAAAAGCGAAAGCTGATACGGGAACAGAGCACGGTTGGAAAGCGATGAGTTACCTAAAGACAATCGGGTACGACTGAGTCGCAATGTTAATCAGATATAAGGTATAAGTTGTGTTTACTGAACGCAAGTTTCTAATTTCGATTATCGTTCGATAGAGGAAAGTGTCTGAAACCTCTAGTACAAAGAAAGGTAAGTTATCCTGCACGACTTATCTGTTATCACCACATTTGTACAATCTG 1542|1543s gBlock TTCCCCTCTAGAAAAAAGCTTATAATTATCCTTACAGCCCTTCCCGGTGCGCCCAGATAGGGTGTTAAGTCAAGTAGTTTAAGGTACTACTCTGTAAGATAACACAGAAAACAGCCAACCTAACCGAAAAGCGAAAGCTGATACGGGAACAGAGCACGGTTGGAAAGCGATGAGTTACCTAAAGACAATCGGGTACGACTGAGTCGCAATGTTAATCAGATATAAGGTATAAGTTGTGTTTACTGAACGCAAGTTTCTAATTTCGATTGGCTGTCGATAGAGGAAAGTGTCTGAAACCTCTAGTACAAAGAAAGGTAAGTTAGCCGGGAAGACTTATCTGTTATCACCACATTTGTACAATCTG 2329|2330a gBlock TTCCCCTCTAGAAAAAAGCTTATAATTATCCTTAGCGGCCTCAGCAGTGCGCCCAGATAGGGTGTTAAGTCAAGTAGTTTAAGGTACTACTCTGTAAGATAACACAGAAAACAGCCAACCTAACCGAAAAGCGAAAGCTGATACGGGAACAGAGCACGGTTGGAAAGCGATGAGTTACCTAAAGACAATCGGGTACGACTGAGTCGCAATGTTAATCAGATATAAGGTATAAGTTGTGTTTACTGAACGCAAGTTTCTAATTTCGATTGCCGCTCGATAGAGGAAAGTGTCTGAAACCTCTAGTACAAAGAAAGGTAAGTTAACTGCTGAGACTTATCTGTTATCACCACATTTGTACAATCTG Primers for gBlock amplification (optional): LtrB-F TTCCCCTCTAGAAAAAAGCTTATAATTATCCTTA LtrB-R Universal primer CAGATTGTACAAATGTGGTGATAACAGATAAGTC Recommended plasmids for TargeTron construction(retargeting): Swap the retargeted fragment of 635|636s TargeTron for the corresponding fragment of pACD4-C donor plasmid. Swap the retargeted fragment of 1063|1064a TargeTron for the corresponding fragment of pACD4-C donor plasmid. Swap the retargeted fragment of 1125|1126s TargeTron for the corresponding fragment of pACD4-G donor plasmid. Swap the retargeted fragment of 1542|1543s TargeTron for the corresponding fragment of pACD4-G donor plasmid. Swap the retargeted fragment of 2329|2330a TargeTron for the corresponding fragment of pACD4-G donor plasmid. TargeTron is a registered trademark of InGex, LLC gBlock is a registered trademark of Integrated DNA Technologies, Inc Copyright TargeTronics, LLC All rights reserved www.targetrons.com