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2. Use this link and choose to design an shRNA, then enter your sequence or accession number.https://rnaidesigner.invitrogen.com/rnaiexpress/setOption.do?designOption=shrna&pid=- (https://rnaidesigner.invitrogen.com/rnaiexpress/setOption.do?designOption=shrna&pid=\-)
248931025050769267

3. Choose hits that are 4-5 stars and blast the sequences to the zebrafish genome to be sure your
target gene is your only hit (anything else should be looked at more carefully since off target effects
are possible depending on your target sequence and region of genome match within the hairpin).

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1     atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
61   ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
121 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
181 ctcgtgacta ccttcggcta cggcctgatg tgcttcgccc gctaccccga ccacatgaag
241 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
301 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
361 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
421 aagctggagt acaactacgg tggatccggt gctagcaaca gccacaacgt ctatatcatg
481 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
541 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
601 ctgctgcccg acaaccacta cctgagctac cagtccgccc tgagcaaaga ccccaacgag
661 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
721 gacgagctgt acaagtaa

pink = 21 mer Invitrogen Block-IT design 5 stars (THIS HAS NO BLAST HITS IN THE ZEBRAFISH
GENOME)

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