Construct Name: pHIPPY GFP22

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Description: This construct is designed for the cloning and expression of siRNAs in mammalian cells. It relies on opposing human H1 and human U6 Pol III promoters to drive expression of siRNAs.


Sequence (note GFP stuffer sequence is lower case): CAATTCGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAG GCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGT GGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGC TCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTC CGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTA GGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCT TGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTG GTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTG AAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTG CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGAT CCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAG CAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTC TACGGGGTCTGACGCTCAGTGGGCGGCCGCATTTGCATGTCGCTATGTGT TCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTAT AAGTGGATCCTGAGACCGTCTCAAAAAgcaagctgaccctgaagttcatT TTTTGAGACGCTAGCCACAAGATATATAAAGCCAAGAAATCGAAATACTT TCAAGTTACGGTAAGCATATGATAGTCCATTTTAAAACATAATTTTAAAA CTGCAAACTACCCAAGAAATTATTACTTTCTACGTCACGTATTTTGTACT AATATCTTTGTGTTTACAGTCAAATTAATTCTAATTATCTCTCTAACAGC CTTGTATCGTATATGCAAATATGAAGGAATCATGGGAAATAGGCCCTCTT CCTGCCCGACCTTGGCGCGCGCTCGGCGCGCGGTCACGCTCCGTCACGTG GTGCGTTTTGCCTGCGCGTCTTTCCACTGGGGACGCGTGGTGTGGAAAGT CCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAG TCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTAT GCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTC CGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCAT GGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTC TGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGTCTCGA TGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACG GATCAAGGAGGTCGACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTC ACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGG GTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACG ACGTGACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAAC ACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTG GTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGA CCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCG GCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGAACTAGCGA TATCACTAGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAG GAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGTGTT GGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGT CGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCT TTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCC AACGTCGGGGCGGCAGG

Notes for Use: Select clones with ZEO (50ug/ml). Vector is 2167 bp. Cloning site for siRNAs is between the BsmBI sites that are presently filled with GFP22siRNA sequence.

Reference: Constructed by Ajamete Kaykas, and available through the Moon lab by standard HHMI MTA agreement. Published reference: A. Kaykas and R. T. Moon, 2004, "A plasmid-based system for expressing small interfering RNA libraries in mammalian cells." BMC Cell Biology 2004, 5:16.