Subject: Flip-out piggyBac transposase constructs and insertions
The following information was provided by Annette Parks of Exelixis, Inc.
In P{FRT(w+)Tub-PBac\T}, the piggyBac transposase coding region with the
3' UTR from fs(1)K10 under the control of the alphaTub84B promoter was
cloned into P{Express-FRT.2.2}. Upon exposure to FLP recombinase, the
miniwhite gene positioned between FRT sites is excised. The rearranged
construct is P{Tub-PBac\T}. Both P{FRT(w+)Tub-PBac\T} and P{Tub-PBac\T}
express piggyBac transposase.
P{FRT(w+)Tub-PBac\T}2 is an insertion on CyO. P{Tub-PBac\T}2 is the same
insertion after excision of the miniwhite cassette by FLP recombinase.
In P{FRT(w+)Hsp70-PBac\T}, the piggyBac transposase coding region under
the control of the Hsp70 promoter was cloned into P{Express-FRT.2.3}. Upon
exposure to FLP recombinase, the miniwhite gene positioned between FRT
sites is excised. The rearranged construct is P{Hsp70-PBac\T}. Both
P{FRT(w+)Hsp70-PBac\T} and P{Hsp70-PBac\T} express piggyBac transposase
upon heat shock.
P{FRT(w+)Hsp70-PBac\T}2 is an insertion on CyO. P{Hsp70-PBac\T}2 is the
same insertion after excision of the miniwhite cassette by FLP recombinase.
The sequence of the alphaTub84B-PBac\T-fs(1)K10 3' UTR insert is given
below and in attached files.
GTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT
GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTT
ATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATC
AGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACG
TTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTC
GGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGA
CAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGG
ACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCG
TTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTG
CGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTG
CAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCG
CGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACT
ATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACT
CATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT
GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGAT
CCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAG
AGCTACCAACTCTTTTT
CCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA
AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTG
TCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAG
CCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAG
GGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGC
CTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG
AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTC
CTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGAC
CGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATT
CATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATG
TGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGG
ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAG
CTGGAGCTCCACCGCGGTGGCGGCCGCTTCGAGTCCCAACTAGTCCTGCAAAATACCGCAGCAAGTTTTAGAGAGACCA
AGTGCCATTACCTCTCCCACTTCAGTTATCGGTTATGCGGCGTTTAAGTCGACAGCTTGCCGTCTCTAGCTCCGGTGCC
TATATAAAGCAGCCCGCTTTCCACATTTCATATTCGTTTTACGTTTGTCAAGCCTCATAGCCGGCAGTTCGAACGTATA
CGCTCTCTGAGTCAGACCTCGAAATCGTAGCTCTACACAATTCTGTGAATTTTCCTTGTCGCGTGTGAAACACTTCCAA
TAAAAACTCAATATGGTGAGTACTTTAAAAAAAAATCTAGTGAAATAATGCTGAAAAGAAATTTGTGTGGGCAAAATTC
AATGGGCAAAAACGCGATGCGGCTTTTTCTCAAAATGGCGGCCGGCCTGCGTTTTTTCCTCAAAAGTGATGACGTCATG
CCTGTTTTTTTTTTTTGTTCGCAATGAGGAATGGCTCTTAAAATCTAGATAAAAAAAATATTCATTATTTCTATGCTGC
TGGAACGCTTCATTAATCTTAAAAATTCTAAATTCGGTTACCATGATACTTCGACGCATAACTGTAGATTTTGGATAGA
ATTAAAGAGAAAATGGCGAGAGAG
TAAAATTCCGGCGTCGGCAAAGTAGAGCAAAAAAATCAGTATACCATTTAGCTACCTCTCTCACTCGCACGCAGTGCCG
GCTCAAGTTGGGCGCGGCTCTGCAATTATCGATTTTCTTGGGGTGTGTAACTAATCATCCGTTTTCCCCTCCTCCTCAT
CCACAGCGTGGGATCCTGGGTAGTTCTTTAGACGATGAGCATATCCTCTCTGCTCTTCTGCAAAGCGATGACGAGCTTG
TTGGTGAGGATTCTGACAGTGAAATATCAGATCACGTAAGTGAAGATGACGTCCAGAGCGATACAGAAGAAGCGTTTAT
AGATGAGGTACATGAAGTGCAGCCAACGTCAAGCGGTAGTGAAATATTAGACGAACAAAATGTTATTGAACAACCAGGT
TCTTCATTGGCTTCTAACAGAATCTTGACCTTGCCACAGAGGACTATTAGAGGTAAGAATAAACATTGTTGGTCAACTT
CAAAGTCCACGAGGCGTAGCCGAGTCTCTGCACTGAACATTGTCAGATCTCAAAGAGGTCCGACGCGTATGTGCCGCAA
TATATATGACCCACTTTTATGCTTCAAACTATTTTTTACTGATGAGATAATTTCGGAAATTGTAAAATGGACAAATGCT
GAGATATCATTGAAACGTCGGGAATCTATGACAGGTGCTACATTTCGTGACACGAATGAAGATGAAATCTATGCTTTCT
TTGGTATTCTGGTAATGACAGCAGTGAGAAAAGATAACCACATGTCCACAGATGACCTCTTTGATCGA
TCTTTGTCAATGGTGTACGTCTCTGTAATGAGTCGTGATCGTTTTGATTTTTTGATACGATGTCTTAGAATGGATGACA
AAAGTATACGGCCCACACTTCGAGAAAACGATGTATTTACTCCTGTTAGAAAAATATGGGATCTCTTTATCCATCAGTG
CATACAAAATTACACTCCAGGGGCTCATTTGACCATAGATGAACAGTTACTTGGTTTTAGAGGACGGTGTCCGTTTAGG
ATGTATATCCCAAACAAGCCAAGTAAGTATGGAATAAAAATCCTCATGATGTGTGACAGTGGTACGAAGTATATGATAA
ATGGAATGCCTTATTTGGGAAGAGGAACACAGACCAACGGAGTACCACTCGGTGAATACTACGTGAAGGAGTTATCAAA
GCCTGTGCACGGTAGTTGTCGTAATATTACGTGTGACAATTGGTTCACCTCAATCCCTTTGGCAAAAAACTTACTACAA
GAACCGTATAAGTTAACCATTGTGGGAACCGTGCGATCAAACAAACGCGAGATACCGGAAGTACTGAAAAACAGTCGCT
CCAGGCCAGTGGGAACATCGATGTTTTGTTTTGACGGACCCCTTACTCTCGTCTCATATAAACCGAAGCCAGCTAAGAT
GGTATACTTATTATCATCTT
GTGATGAGGATGCTTCTATCAACGAAAGTACCGGTAAACCGCAAATGGTTATGTATTATAATCAAACTAAAGGCGGAGT
GGACACGCTAGACCAAATGTGTTCTGTGATGACCTGCAGTAGGAAGACGAATAGGTGGCCTATGGCATTATTGTACGGA
ATGATAAACATTGCCTGCATAAATTCTTTTATTATATACAGCCATAATGTCAGTAGCAAGGGAGAAAAGGTCCAAAGTC
GCAAAAAATTTATGAGAAACCTTTACATGAGCCTGACGTCATCGTTTATGCGTAAGCGTTTAGAAGCTCCTACTTTGAA
GAGATATTTGCGCGATAATATCTCTAATATTTTGCCAAATGAAGTGCCTGGTACATCAGATGACAGTACTGAAGAGCCA
GTAATGAAAAAACGTACTTACTGTACTTACTGCCCCTCTAAAATAAGGCGAAAGGCAAATGCATCGTGCAAAAAATGCA
AAAAAGTTATTTGTCGAGAGCATAATATTGATATGTGCCAAAGTTGTTTCTGACTGACTAATAAGTATAATTTGTTTCT
ATTATGTATAAGTTAAGCTAATTACTTATTTTATAATACAACATGACTGTTTTTAAAGTACAAAA
TAAGTTTATTTTTGTAAAAGAGAGAATGTTTAAAAGTTTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTT
TAATAAATAAATAAACATAAATAAATTGTTTGTTGAATTTATTATTAGTATGTAAGTGTAAATATAATAAAACTTAATA
TCTATTCAAATTAATAAATAAACCTCGATATACAGACCGATAAAACACATGCGTCAATTTTACGCATGATTATCTTTAA
CGTACGAGGCCTTAGATTACACCACTTGATTGTATTTTTAAATTAATTCTTAAAAACTACAAATTAAGATCACTCTGTG
AACGTGTGCTCGATGGTGTGCATCTACGGATTTGTTTTTGTGTTCTTTTTCCCCACCCAACCCCGTCCCTGACCCAATT
CCGCTGTTCTCTTTACCGACTTGTAGATTAATCACGCTCTTGATCTTCATGACCGGATCCACCTGACAAGTAACGCCAA
AGCAGCCCCACTTAG
CGGAATAGTAGAGCCTAGGGGACCCAACGTAAAAACTCTAGTATAGCACCAATCTCCCAACCAGTTAACATTATACCTA
AACCCATGGTCAAGAGTAAACATTTCTGCCTTTGAAGTTGAGAACACAATTAAGCATCCCCTGGTTAAACCTGACATTC
ATACTTGTTAATAGCGCCATAAACATAGCACCAATTTCGAAGAAATCAGTTAAAAGCAATTAGCAATTAGCAATTAGCA
ATAACTCTGCTGACTTCAAAACGAGAAGAGTTACAAGTATTTGTAAGGCACAGTTTATAGACCACCGACGGCTCGTTAG
GGCTCGTCATGTAACTAAGCGCGGTGAAACCCAATTGAACATATAGTGGAATTATTATTATCAATGGGGAATATTTAAC
CCTCAGGTAGCAAAGTAATTTAATTGCAAATAGAGAGTCCTAAGACTAAATAATATATTTAAAAATCTGGCCCTTTGAC
CTTGCTTGTCAGGTGCATTTGGGTTCAATCGTAAGTTGCTTCTATATAAACACTTTCCCCATCCCCGCAATAATGAAGA
ATACCGCAGAATAAAGAGAGATTTGCAACAAAAAATAAAGGCATTGCGAAAACTTTTTATGGGGGATCATTACACTCGG
GCCTACGGTTACAATTCCCAGCCACTTAAGCGACAAGTTTGGCCAACAATCCATCTAATAGCTAATAGCGCAATCACTG
GTAATCGCAAGAGTATATAGGCAATAGAACCCATGGATTTGCCCAAAGGTAACCGAGACAATGGAGAAGCAAGAGGATT
TCAAACTGAACACCCACAGTGCTGTGTACTACCACTGGCGCGTTTGGGAGCTCACTGG
CCTGATGCGTCCTCCGGGCGTTTCAAGCCTGCTTTACGTGGTATACTCCATTACGGTCAACTTGGTGGTCACCGTGCTG
TTTCCCTTGAGCTTGCTGGCCAGGCTGCTGTTCACCACCAACATGGCCGGATTGTGCGAGAACCTGACCATAACTATTA
CCGATATTGTGGCCAATTTGAAGTTTGCGAATGTGTACATGGTGAGGAAGCAGCTCCATGAGATTCGCTCTCTCCTAAG
GCTCATGGACGCTAGAGCCCGGCTGGTGGGCGATCCCGAGGAGATTTCTGCCTTGAGGAAGGAAGTGAATATCGCACAG
GGCACTTTCCGCACCTTTGCCAGTATTTTCGTATTTGGCACTACTTTGAGTTGCGTCCGCGTGGTCGTTCGCCCGGATC
GAGAGCTCCTGTATCCGGCCTGGTTCGGCGTTGACTGGATGCACTCCACCAGAAACTATGTGCTCATCAATATCTACCA
GCTCTTCGGCTTGATAGTGCAGGCTATACAGAACTGCGCTAGTGACTCCTATCCGCCTGCGTTTCTCTGCCTGCTCACG
GGTCATATGCGTGCTTTGGAGCTGAGGGTGCGGCGGATTGGCTGCAGGACGGAAAAGTCCAATAAAGGGC
AGACGGTACCAAGCTTGATCGATACCGTCGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCTATAGTGAGTCGTATT
ACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGC
ACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAAT
GGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTG
CCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCT
AAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGT
TCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCT
TGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA
TTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAG