EcI5 TargeTron Design December 02 2012 Predicted insertion sites for lacZ: -1|+1 -30 -1 EcI5 +1 +15 score 163|164s CCGATCGCCCTTCCCAACAGTTGCGCAGCC gtgc...cgat TGAATGGCGAATGGC 11.7 912|913s AACGTCGAAAACCCGAAACTGTGGAGCGCC gtgc...cgat GAAATCCCGAATCTC 10.6 1257|1258s GTGGTGGATGAAGCCAATATTGAAACCCAC gtgc...cgat GGCATGGTGCCAATG 9.9 1806|1807s TTTGGCGATACGCCGAACGATCGCCAGTTC gtgc...cgat TGTATGAACGGTCTG 11.0 2427|2428s ACCCGCATTGACCCTAACGCCTGGGTCGAA gtgc...cgat CGCTGGAAGGCGGCG 11.2 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: 163|164s IBS1/2S CCCCTCTAGAAGAATTCCCATGCCAAACAGTTGCGCAGCCGTGCGACATGAAGTCG 163|164s EBS1S CAGGCTTGAACCAAAAGGTATGTGGTTGGTTACTCCTCTGGCTGCTAGGGGTACACGGAC 163|164s EBS2AS TACCTTTTGGTTCAAGCCTGTCAGCATCTTTGGCTTGTTCAGTTTAACGACGCTTCAGC 912|913s IBS1/2S CCCCTCTAGAAGAATTCCCATGCCAAAACTGTGGAGCGCCGTGCGACATGAAGTCG 912|913s EBS1S CAGGCTTGAACCAAAAGGTATGTGGTTGGTTACTCCTCTGGCGCCTAGGGGTACACGGAC 912|913s EBS2AS TACCTTTTGGTTCAAGCCTGTCAGCATCTTTGGCTTGTTACTGTTAACGACGCTTCAGC 1257|1258s IBS1/2S CCCCTCTAGAAGAATTCCCATGCCAAATATTGAAACCCACGTGCGACATGAAGTCG 1257|1258s EBS1S CAGGCTTGAACCAAAAGGTATGTGGTTGGTTACTCCTCTGTGGGCTAGGGGTACACGGAC 1257|1258s EBS2AS TACCTTTTGGTTCAAGCCTGTCAGCATCTTTGGCTTGTTTATTGTAACGACGCTTCAGC 1806|1807s IBS1/2S CCCCTCTAGAAGAATTCCCATGCCAAACGATCGCCAGTTCGTGCGACATGAAGTCG 1806|1807s EBS1S CAGGCTTGAACCAAAAGGTATGTGGTTGGTTACTCCTCTGAACTCTAGGGGTACACGGAC 1806|1807s EBS2AS TACCTTTTGGTTCAAGCCTGTCAGCATCTTTGGCTTGTTCGATCTAACGACGCTTCAGC 2427|2428s IBS1/2S CCCCTCTAGAAGAATTCCCATGCCAAACGCCTGGGTCGAAGTGCGACATGAAGTCG 2427|2428s EBS1S CAGGCTTGAACCAAAAGGTATGTGGTTGGTTACTCCTCTTTCGACTAGGGGTACACGGAC 2427|2428s EBS2AS TACCTTTTGGTTCAAGCCTGTCAGCATCTTTGGCTTGTTCGCCTTAACGACGCTTCAGC EBSR Universal primer TATCCGGTCCATTACAGACTGGCATTC Recommended primers and plasmids for the EcI5-EBS template preparation: Use pACD3-EcI5A donor plasmid and primer EcI5-EBS-A-F with EcI5-EBS-R to make the EcI5-EBS template for 163|164s TargeTron. Use pACD3-EcI5C donor plasmid and primer EcI5-EBS-C-F with EcI5-EBS-R to make the EcI5-EBS template for 912|913s TargeTron. Use pACD3-EcI5C donor plasmid and primer EcI5-EBS-C-F with EcI5-EBS-R to make the EcI5-EBS template for 1257|1258s TargeTron. Use pACD3-EcI5A donor plasmid and primer EcI5-EBS-A-F with EcI5-EBS-R to make the EcI5-EBS template for 1806|1807s TargeTron. Use pACD3-EcI5G donor plasmid and primer EcI5-EBS-G-F with EcI5-EBS-R to make the EcI5-EBS template for 2427|2428s TargeTron. gBlocks Gene Fragments (have to be extended by PCR) to modify the TargeTron for insertion into predicted sites: 163|164s gBlock CCCCTCTAGAAGAATTCCCATGCCAAACAGTTGCGCAGCCGTGCGACATGAAGTCGCCTGAATAATTGTTCCAGCGGAGTTCGATTCCGTCAGGGAACCTGATGTTCCGTCATCAGTAGCCTACTGACACATGCGTCACTGGTAACGGTGGGGTGTGAAGCTGTCAGGACAATGAAACCGGATCTTCGGATCGCATGAAACCGTGAGGTTACATGTAATCTGCCAGCATCAGGCGGAGGAGGTCTAGGCTCGGTAGCATGACTAACATATGTGAACTGCTGAAGCGTCGTTAAACTGAACAAGCCAAAGATGCTGACAGGCTTGAACCAAAAGGTATGTGGTTGGTTACTCCTCTGGCTGCTAGGGGTACACGGACAATAAACCACCGGTGTTATGAGCAGAGTCTAACCTACTGTTGTTATTCAGGTGGAACATGGTAAGCCCGTATCGCTGCCTCGTGAGGCAGGTTAACCGCAAGGGATACTGTTGGCGGTGCGGGT 912|913s gBlock CCCCTCTAGAAGAATTCCCATGCCAAAACTGTGGAGCGCCGTGCGACATGAAGTCGCCTGAATAATTGTTCCAGCGGAGTTCGATTCCGTCAGGGAACCTGATGTTCCGTCATCAGTAGCCTACTGACACATGCGTCACTGGTAACGGTGGGGTGTGAAGCTGTCAGGACAATGAAACCGGATCTTCGGATCGCATGAAACCGTGAGGTTACATGTAATCTGCCAGCATCAGGCGGAGGAGGTCTAGGCTCGGTAGCATGACTAACATATGTGAACTGCTGAAGCGTCGTTAACAGTAACAAGCCAAAGATGCTGACAGGCTTGAACCAAAAGGTATGTGGTTGGTTACTCCTCTGGCGCCTAGGGGTACACGGACAATAAACCACCGGTGTTCTGAGCAGAGTCTAACCTACTGTTGTTATTCAGGTGGAACATGGTAAGCCCGTATCGCTGCCTCGTGAGGCAGGTTAACCGCAAGGGATACTGTTGGCGGTGCGGGT 1257|1258s gBlock CCCCTCTAGAAGAATTCCCATGCCAAATATTGAAACCCACGTGCGACATGAAGTCGCCTGAATAATTGTTCCAGCGGAGTTCGATTCCGTCAGGGAACCTGATGTTCCGTCATCAGTAGCCTACTGACACATGCGTCACTGGTAACGGTGGGGTGTGAAGCTGTCAGGACAATGAAACCGGATCTTCGGATCGCATGAAACCGTGAGGTTACATGTAATCTGCCAGCATCAGGCGGAGGAGGTCTAGGCTCGGTAGCATGACTAACATATGTGAACTGCTGAAGCGTCGTTACAATAAACAAGCCAAAGATGCTGACAGGCTTGAACCAAAAGGTATGTGGTTGGTTACTCCTCTGTGGGCTAGGGGTACACGGACAATAAACCACCGGTGTTCTGAGCAGAGTCTAACCTACTGTTGTTATTCAGGTGGAACATGGTAAGCCCGTATCGCTGCCTCGTGAGGCAGGTTAACCGCAAGGGATACTGTTGGCGGTGCGGGT 1806|1807s gBlock CCCCTCTAGAAGAATTCCCATGCCAAACGATCGCCAGTTCGTGCGACATGAAGTCGCCTGAATAATTGTTCCAGCGGAGTTCGATTCCGTCAGGGAACCTGATGTTCCGTCATCAGTAGCCTACTGACACATGCGTCACTGGTAACGGTGGGGTGTGAAGCTGTCAGGACAATGAAACCGGATCTTCGGATCGCATGAAACCGTGAGGTTACATGTAATCTGCCAGCATCAGGCGGAGGAGGTCTAGGCTCGGTAGCATGACTAACATATGTGAACTGCTGAAGCGTCGTTAGATCGAACAAGCCAAAGATGCTGACAGGCTTGAACCAAAAGGTATGTGGTTGGTTACTCCTCTGAACTCTAGGGGTACACGGACAATAAACCACCGGTGTTATGAGCAGAGTCTAACCTACTGTTGTTATTCAGGTGGAACATGGTAAGCCCGTATCGCTGCCTCGTGAGGCAGGTTAACCGCAAGGGATACTGTTGGCGGTGCGGGT 2427|2428s gBlock CCCCTCTAGAAGAATTCCCATGCCAAACGCCTGGGTCGAAGTGCGACATGAAGTCGCCTGAATAATTGTTCCAGCGGAGTTCGATTCCGTCAGGGAACCTGATGTTCCGTCATCAGTAGCCTACTGACACATGCGTCACTGGTAACGGTGGGGTGTGAAGCTGTCAGGACAATGAAACCGGATCTTCGGATCGCATGAAACCGTGAGGTTACATGTAATCTGCCAGCATCAGGCGGAGGAGGTCTAGGCTCGGTAGCATGACTAACATATGTGAACTGCTGAAGCGTCGTTAAGGCGAACAAGCCAAAGATGCTGACAGGCTTGAACCAAAAGGTATGTGGTTGGTTACTCCTCTTTCGACTAGGGGTACACGGACAATAAACCACCGGTGTTGTGAGCAGAGTCTAACCTACTGTTGTTATTCAGGTGGAACATGGTAAGCCCGTATCGCTGCCTCGTGAGGCAGGTTAACCGCAAGGGATACTGTTGGCGGTGCGGGT gBlock extension is required because the max. length of a gBlock is 500 bp. Retargeted fragment of the EcI5 TargeTron is larger than 500 bp. Primers for gBlock extension (required): EcI5-F CCCCTCTAGAAGAATTCCCATGCCAAA EcI5-R Universal primer ATCCGGTCCATTACAGACTGGCATTCGCTTTTTCCACCTTCTTTTACCCGCACCGCCAAC Recommended plasmids for TargeTron construction(retargeting): Swap the retargeted fragment of 163|164s TargeTron for the corresponding fragment of pACD3-EcI5A donor plasmid. Swap the retargeted fragment of 912|913s TargeTron for the corresponding fragment of pACD3-EcI5C donor plasmid. Swap the retargeted fragment of 1257|1258s TargeTron for the corresponding fragment of pACD3-EcI5C donor plasmid. Swap the retargeted fragment of 1806|1807s TargeTron for the corresponding fragment of pACD3-EcI5A donor plasmid. Swap the retargeted fragment of 2427|2428s TargeTron for the corresponding fragment of pACD3-EcI5G donor plasmid. 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