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Contents:In the table below, Illumina Nextera Handle sequences have been added and highlighted in bold. Primers for the on-target have been added for convenience. The table below is sorted by the CFD off-target score. Sites with very low CFD scores < 0.02 are unlikely to be cleaved, see our study Haeussler et al. 2016, Figure 2.
In the protocol by Matthew Canver, Harvard, two PCRs are run: one PCR to amplify the potential off-target, then a second PCR to extend the handles with Illumina barcodes. Please click here to download the protocol. Alternatively, you can have a look at Fu et al, 2014.
If a primer was not found, the reason is usually that the region around the off-target is too repetitive. To avoid unspecific primers, all repeats are masked for the primer design (not for off-target search). If you think that we should change the parameters here or should use different primer3 settings, please let us know.
Name | Primer Sequence | Tm | CFD Score |
---|---|---|---|
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313686_F | TCGTCGGCAGCGTCCGCATCACAAGGTGACCCTA | 59.7 | 1.00 |
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313686_R | GTCTCGTGGGCTCGGCTCCTAATCCCAGGCTCCCT | 60.1 | 1.00 |
mm4_intergenic_Ap4e1|Gm24081_chr2_127039241_F | Primer3: not found at this Tm | N.d. | 0.40 |
mm4_intergenic_Ap4e1|Gm24081_chr2_127039241_R | Primer3: not found at this Tm | N.d. | 0.40 |
mm4_intron_Hhat_chr1_192731348_F | Primer3: not found at this Tm | N.d. | 0.36 |
mm4_intron_Hhat_chr1_192731348_R | Primer3: not found at this Tm | N.d. | 0.36 |
mm4_intergenic_Gm23673|Gm9381_chr3_124955989_F | TCGTCGGCAGCGTCTCCTGCTCTCCTCTACAACCA | 59.9 | 0.29 |
mm4_intergenic_Gm23673|Gm9381_chr3_124955989_R | GTCTCGTGGGCTCGGAGTGTTCCCAGAAGGATGTCA | 58.6 | 0.29 |
mm4_intergenic_Mir5112|4930445N18Rik_chr18_82839190_F | TCGTCGGCAGCGTCGCATTGTTTGGGCCACCTTC | 60.3 | 0.26 |
mm4_intergenic_Mir5112|4930445N18Rik_chr18_82839190_R | GTCTCGTGGGCTCGGGCCCATTAAGACAGAGCTCCA | 59.7 | 0.26 |
mm4_intergenic_Arnt2|Gm25790_chr7_84314438_F | Primer3: not found at this Tm | N.d. | 0.26 |
mm4_intergenic_Arnt2|Gm25790_chr7_84314438_R | Primer3: not found at this Tm | N.d. | 0.26 |
mm4_intron_Gcnt1_chr19_17336816_F | TCGTCGGCAGCGTCCCAAGCTCAGCTCTGACACA | 59.9 | 0.21 |
mm4_intron_Gcnt1_chr19_17336816_R | GTCTCGTGGGCTCGGACCGCTTTGCAGTGTTTTCA | 59.1 | 0.21 |
mm4_exon_mmu-mir-7667_chr17_29595579_F | Primer3: not found at this Tm | N.d. | 0.18 |
mm4_exon_mmu-mir-7667_chr17_29595579_R | Primer3: not found at this Tm | N.d. | 0.18 |
mm4_intergenic_Ralgps2|Tex35_chr1_157073106_F | Primer3: not found at this Tm | N.d. | 0.18 |
mm4_intergenic_Ralgps2|Tex35_chr1_157073106_R | Primer3: not found at this Tm | N.d. | 0.18 |
mm3_intron_Gcnt4_chr13_96944696_F | TCGTCGGCAGCGTCCCCTTGGCCTGTGCTTCTAA | 59.9 | 0.14 |
mm3_intron_Gcnt4_chr13_96944696_R | GTCTCGTGGGCTCGGAACTGCAGATCCTGATGGCC | 60.1 | 0.14 |
mm4_intergenic_1110036E04Rik|Scarletltr_chr9_64079571_F | Primer3: not found at this Tm | N.d. | 0.10 |
mm4_intergenic_1110036E04Rik|Scarletltr_chr9_64079571_R | Primer3: not found at this Tm | N.d. | 0.10 |
mm4_intergenic_Zfp652|Phospho1_chr11_95795297_F | Primer3: not found at this Tm | N.d. | 0.09 |
mm4_intergenic_Zfp652|Phospho1_chr11_95795297_R | Primer3: not found at this Tm | N.d. | 0.09 |
mm4_exon_Scx_chr15_76458080_F | TCGTCGGCAGCGTCCTTTCTTCCACAGCGGTCGT | 60.6 | 0.08 |
mm4_exon_Scx_chr15_76458080_R | GTCTCGTGGGCTCGGAGATCTGTTTGGGCTGGGTG | 59.9 | 0.08 |
mm3_intergenic_C530025M09Rik/Syndig1|Syndig1_chr2_149831400_F | TCGTCGGCAGCGTCTAGCTGAGTCCAAGAGGGGC | 60.9 | 0.08 |
mm3_intergenic_C530025M09Rik/Syndig1|Syndig1_chr2_149831400_R | GTCTCGTGGGCTCGGGCTTTCCTGTCCCATGTGGA | 59.9 | 0.08 |
mm4_exon_Gm20421/Hes5_chr4_154961384_F | TCGTCGGCAGCGTCACTCATGCCGCTTCGTCTC | 60.1 | 0.07 |
mm4_exon_Gm20421/Hes5_chr4_154961384_R | GTCTCGTGGGCTCGGAGGAACTGTACCGCCTCCT | 59.9 | 0.07 |
mm4_intergenic_Gm11340|Gm23340_chr13_24291955_F | Primer3: not found at this Tm | N.d. | 0.07 |
mm4_intergenic_Gm11340|Gm23340_chr13_24291955_R | Primer3: not found at this Tm | N.d. | 0.07 |
mm4_intron_Agap1_chr1_89647258_F | Primer3: not found at this Tm | N.d. | 0.07 |
mm4_intron_Agap1_chr1_89647258_R | Primer3: not found at this Tm | N.d. | 0.07 |
mm4_exon_Opa3_chr7_19228430_F | Primer3: not found at this Tm | N.d. | 0.06 |
mm4_exon_Opa3_chr7_19228430_R | Primer3: not found at this Tm | N.d. | 0.06 |
mm4_intron_Wdfy3_chr5_101995880_F | Primer3: not found at this Tm | N.d. | 0.05 |
mm4_intron_Wdfy3_chr5_101995880_R | Primer3: not found at this Tm | N.d. | 0.05 |
mm4_intergenic_Gm26260|4930564G21Rik_chr10_119645796_F | TCGTCGGCAGCGTCAATGCCCCTGTAAAGCACCT | 59.5 | 0.04 |
mm4_intergenic_Gm26260|4930564G21Rik_chr10_119645796_R | GTCTCGTGGGCTCGGCATTTCCCACGATGACCCCA | 60.0 | 0.04 |
mm4_intergenic_2610028E06Rik|Csf3r_chr4_125977061_F | TCGTCGGCAGCGTCCACCATCTCGCTCCTTCAGG | 60.1 | 0.04 |
mm4_intergenic_2610028E06Rik|Csf3r_chr4_125977061_R | GTCTCGTGGGCTCGGGTTGAGGTGAGGGTCATGGG | 60.0 | 0.04 |
mm4_exon_4933400F03Rik/Kcnmb4_chr10_116473829_F | Primer3: not found at this Tm | N.d. | 0.04 |
mm4_exon_4933400F03Rik/Kcnmb4_chr10_116473829_R | Primer3: not found at this Tm | N.d. | 0.04 |
mm4_exon_Phf15_chr11_51857599_F | TCGTCGGCAGCGTCCTCCATGGACGCTACTCACC | 59.8 | 0.04 |
mm4_exon_Phf15_chr11_51857599_R | GTCTCGTGGGCTCGGCCCCTCTAAAGAGATCACAAGGA | 58.9 | 0.04 |
mm4_intergenic_Cdc7|Tgfbr3_chr5_107093819_F | TCGTCGGCAGCGTCTGAGCAGCCTGACTTTGAGG | 59.9 | 0.02 |
mm4_intergenic_Cdc7|Tgfbr3_chr5_107093819_R | GTCTCGTGGGCTCGGTCTGAGAGGGGTGACTGGAG | 59.9 | 0.02 |
mm4_intergenic_Zdhhc22|Tmem63c_chr12_86990587_F | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intergenic_Zdhhc22|Tmem63c_chr12_86990587_R | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intron_Col27a1_chr4_63220086_F | Primer3: not found at this Tm | N.d. | 0.02 |
mm4_intron_Col27a1_chr4_63220086_R | Primer3: not found at this Tm | N.d. | 0.02 |
mm3_intron_Siah3_chr14_75456210_F | TCGTCGGCAGCGTCAACCCCACGCACAACCTAAA | 60.1 | 0.02 |
mm3_intron_Siah3_chr14_75456210_R | GTCTCGTGGGCTCGGCAGCAAACTGCCTGCATGAG | 60.1 | 0.02 |
mm4_intron_Nsg1_chr5_38159111_F | TCGTCGGCAGCGTCCACAGCCAGAAGGACTCCG | 60.0 | 0.01 |
mm4_intron_Nsg1_chr5_38159111_R | GTCTCGTGGGCTCGGGCCTGAGACTCTGATCTGGC | 59.8 | 0.01 |
mm4_exon_Mier1_chr4_103119401_F | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_exon_Mier1_chr4_103119401_R | Primer3: not found at this Tm | N.d. | 0.01 |
mm4_intergenic_Il34|Gm15895/Il34_chr8_110792033_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Il34|Gm15895/Il34_chr8_110792033_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Mdga1_chr17_29888672_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Mdga1_chr17_29888672_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Gm12688|Foxd3/Gm23366_chr4_99656190_F | TCGTCGGCAGCGTCGGCCAGTCCTTAAAACGGGA | 59.9 | 0.00 |
mm4_intergenic_Gm12688|Foxd3/Gm23366_chr4_99656190_R | GTCTCGTGGGCTCGGCTCCAAGCAGCTCTGGAGG | 59.7 | 0.00 |
mm4_intron_Map7d1_chr4_126237679_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Map7d1_chr4_126237679_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Gm26531_chr12_85977852_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intron_Gm26531_chr12_85977852_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Slc22a30|Gm6425_chr19_8407849_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Slc22a30|Gm6425_chr19_8407849_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Slc22a28|Slc22a29_chr19_8134709_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Slc22a28|Slc22a29_chr19_8134709_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Gm15387|Bai1_chr15_74407438_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Gm15387|Bai1_chr15_74407438_R | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Gm12825|Skint11_chr4_114117285_F | Primer3: not found at this Tm | N.d. | 0.00 |
mm4_intergenic_Gm12825|Skint11_chr4_114117285_R | Primer3: not found at this Tm | N.d. | 0.00 |
These only list off-targets that have primers in the table above. Primers underlined, off-targets in bold.
ontarget_mm0_exon_5730457N03Rik/Evx1_chr6_52313686 |
CGCATCACAAGGTGACCCTAGCTCCCCACTGCCATCTCCGCGGTCGCCGTCGACACGGCGCTGGGGCTACCCGGCGCCTG CCTTGTCGCCTTAGCTCCTCTTCTCAGCCAAGATCCCAGGGAGCCTGGGATTAGGAG |
mm4_intergenic_Gm23673|Gm9381_chr3_124955989 |
TCCTGCTCTCCTCTACAACCACCATCAGGCTCTAAACAACAGTGACAGCTGAGGTGGCAGTGGGATTTCACATGTACATG CAGACCAAAAAAACTCCAACCTGTGTTTTGTACTTTGACATCCTTCTGGGAACACT |
mm4_intergenic_Mir5112|4930445N18Rik_chr18_82839190 |
GCATTGTTTGGGCCACCTTCTTTCTTATCTTGATGGGCGGGGCCACGGAGAAGGCAGTGGGGCCTTTTCGTTTAAGTGTC CTTTCTAGATCTGGGTGAGGATATCTACCTGTGGAGCTCTGTCTTAATGGGC |
mm4_intron_Gcnt1_chr19_17336816 |
CCAAGCTCAGCTCTGACACAGAGAGTAAATCCCAGAACTGTGACAGAGCAGATGGCAGTAGGAAAGGCCCCAAGAGGGAT GTGTTCACAAAGTTCAATGGACCGGAGTGAAAACACTGCAAAGCGGT |
mm3_intron_Gcnt4_chr13_96944696 |
CCCTTGGCCTGTGCTTCTAAATCCCATTCCCTGAGAGCACATCTCAGCCTTCTGTCTGTGGTTGCCTGAGGTCCTTTTCT CAGCTCACTGCCATCTCCGTGCTCACCCAGGCCATCAGGATCTGCAGTT |
mm4_exon_Scx_chr15_76458080 |
CTTTCTTCCACAGCGGTCGTGCGGGCAGCCCACTGCCACCGCCGCCGCCGCCACCACCACTGGCCAGAGACGGCGGCGAG AACACCCAGCCCAAACAGATCT |
mm3_intergenic_C530025M09Rik/Syndig1|Syndig1_chr2_149831400 | TAGCTGAGTCCAAGAGGGGCTGGCGAGAAAGGCATCCGCGGAGGTGGCAGTCAGTGAGGTCCACATGGGACAGGAAAGC |
mm4_exon_Gm20421/Hes5_chr4_154961384 |
ACTCATGCCGCTTCGTCTCCCCACAGCCTTCGCCGCGGCCGCCGGCCCCAAGAGCCTGCACCAGGACTACAGCGAGGGCT ACTCCTGGTGCCTGCAGGAGGCGGTACAGTTCCT |
mm4_intergenic_Gm26260|4930564G21Rik_chr10_119645796 |
AATGCCCCTGTAAAGCACCTAGAAGATTCATTATTGATCTGTGTTTTATTCCTACTTCCAGCTCCGGGGTCTCAGGAATC CTCACTACAGCCGCTCTGGGGTCATCGTGGGAAATG |
mm4_intergenic_2610028E06Rik|Csf3r_chr4_125977061 |
CACCATCTCGCTCCTTCAGGCCTCTGCCCCCCACCTGCCATCTCCTAGGTCCCTGACTATACTCATTATGCTGGAGCCCC TGAGGCAGGCCTAGGATCTGCAGCTACCCATGACCCTCACCTCAAC |
mm4_exon_Phf15_chr11_51857599 |
CTCCATGGACGCTACTCACCCCCCAAAAAAATAGCCTCCAACTGCGATGGCCGCGGTCGGCACCATTCACCAACCCCCTG TGTATGTTTCTGTTTCTTTTTAAACCAAGACTTCCTTGTGATCTCTTTAGAGGGG |
mm4_intergenic_Cdc7|Tgfbr3_chr5_107093819 |
TGAGCAGCCTGACTTTGAGGAAAAAAGTCGGCAGAGAGCCCCAGTCTCCAAAACTCCTAGATCTGGGGGACAGTCCAGTG CCCACTGCATTCTCCCCGGTGGCTCTCTAACATAATAGGGCTCCAGTCACCCCTCTCAGA |
mm3_intron_Siah3_chr14_75456210 |
AACCCCACGCACAACCTAAAGGTGAGTCGCTTCCTCTCGTGCTTTCTGCCATCTCCGCGGGGGCTCTAGCCTTTTCTCAG GAGACTTCTATCAGGGAGCTCATGCAGGCAGTTTGCTG |
mm4_intron_Nsg1_chr5_38159111 |
CACAGCCAGAAGGACTCCGCACACCCACCCGCTGTCTCCGCGGTCGCTGTCCCCGCCTTATCTCCCCAGAGAGGTAGCAG GGACGCGGCGATCTAGCCAGATCAGAGTCTCAGGC |
mm4_intergenic_Gm12688|Foxd3/Gm23366_chr4_99656190 |
GGCCAGTCCTTAAAACGGGACTTTCGACTACCCAGGCTTGGCTTCCCTGTCACCACCCCCCCAACCCCCAACCCCGCGGT CGCCCATGCCCGCGCCCTCCAGAGCTGCTTGGAG |