Epgfp: Difference between revisions
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== | == EPGFPII == | ||
[[image:Small_map.jpg||left|700x700px|EpGFPII plasmid vector]] | [[image:Small_map.jpg||left|700x700px|EpGFPII plasmid vector]] | ||
Revision as of 14:59, 16 April 2020
The plasmid vector used as a vehicle to test the cis-regulatory function of randomly cloned genomic DNA fragments was modified from a previous construct, EpGFP (Arnone et al., 1997; Arnone et al., 1998). The grand-parent of this plasmid was pCMV (Clontech); it has ampicillin resistance. EpGFP contains the region around the start of transcription of the endo16 gene (from -117 to +20).The activity of this element has been described in detail elsewhere (Yuh and Davidson, 1996; Yuh et al., 1996,1998, 2001). In addition to a TATA-containing basal promoter, it contains auxiliary sites for a ubiquitous sea urchin transcription factor which promotes DNA looping and has weak transcriptional enhancement activity(GCF1; Zeller et al., 1995). The vector also includes a sea urchin ribosome binding site from the cyIIa gene (Arnone et al., 1998), positioned at the start of the coding sequence of the GFP (S65T) reporter. The original EpGFP construct was further modified by the addition of a double-stranded oligonucleotide polylinker inserted between the MluI and XmaI sites in the Multiple Cloning Site (MCS), so that the MCS contains the following restriction enzyme sites: 5'-KpnI-SacI-MluI-EcoRI-SpeI-BglII-SmaI/XmaI-3', situated just upstream of the endo16 basal promoter.
EPGFPII
EpGFPII FASTA Sequence
1 11 21 31 41 51 61 71 81 90 | | | | | | | | | | GGTACCGAGC TCTTACGCGT GAATTCACTA GTAGATCTCC CGGGTTAAAC TGTTTGAGTT TCGTCTCCTG ATTGTGCTAT CAAAGACAAA 90 epgfpii GGGGTGTAAC TTTACCCCCC TCATCAAGAG CGGAGGGTTA AATAGAGAAA GACTGGTCGA GGACAGGTCA TAATATTGCT AATTTTTAAG 180 epgfpii CTTATCATCA TGTGTGACGA CGACGTCGCC GCTCTTGTCG TCGACAACGG ATCTGCGGCC GCCGCCACCA TGAGCAAGGG CGAGGAACTG 270 epgfpii TTCACTGGCG TGGTCCCAAT TCTCGTGGAA CTGGATGGCG ATGTGAATGG GCACAAATTT TCTGTCAGCG GAGAGGGTGA AGGTGATGCC 360 epgfpii ACATACGGAA AGCTCACCCT GAAATTCATC TGCACCACTG GAAAGCTCCC TGTGCCATGG CCAACACTGG TCACTACCTT CACCTATGGC 450 epgfpii GTGCAGTGCT TTTCCAGATA CCCAGACCAT ATGAAGCAGC ATGACTTTTT CAAGAGCGCC ATGCCCGAGG GCTATGTGCA GGAGAGAACC 540 epgfpii ATCTTTTTCA AAGATGACGG GAACTACAAG ACCCGCGCTG AAGTCAAGTT CGAAGGTGAC ACCCTGGTGA ATAGAATCGA GCTGAAGGGC 630 epgfpii ATTGACTTTA AGGAGGATGG AAACATTCTC GGCCACAAGC TGGAATACAA CTATAACTCC CACAATGTGT ACATCATGGC CGACAAGCAA 720 epgfpii AAGAATGGCA TCAAGGTCAA CTTCAAGATC AGACACAACA TTGAGGATGG ATCCGTGCAG CTGGCCGACC ATTATCAACA GAACACTCCA 810 epgfpii ATCGGCGACG GCCCTGTGCT CCTCCCAGAC AACCATTACC TGTCCACCCA GTCTGCCCTG TCTAAAGATC CCAACGAAAA GAGAGACCAC 900 epgfpii ATGGTCCTGC TGGAGTTTGT GACCGCTGCT GGGATCACAC ATGGCATGGA CGAGCTGTAC AAGTGAGCGG CCGCGGCTCG AGGCTCTAGA 990 epgfpii GTCGGGGCGG CCGGCCGCTT CGAGCAGACA TGATAAGATA CATTGATGAG TTTGGACAAA CCACAACTAG AATGCAGTGA AAAAAATGCT 1080 epgfpii TTATTTGTGA AATTTGTGAT GCTATTGCTT TATTTGTAAC CATTATAAGC TGCAATAAAC AAGTTAACAA CAACAATTGC ATTCATTTTA 1170 epgfpii TGTTTCAGGT TCAGGGGGAG GTGTGGGAGG TTTTTTAAAG CAAGTAAAAC CTCTACAAAT GTGGTAAAAT CGATAAGGAT CCGTCGACCG 1260 epgfpii ATGCCCTTGA GAGCCTTCAA CCCAGTCAGC TCCTTCCGGT GGGCGCGGGG CATGACTATC GTCGCCGCAC TTATGACTGT CTTCTTTATC 1350 epgfpii ATGCAACTCG TAGGACAGGT GCCGGCAGCG CTCTTCCGCT TCCTCGCTCA CTGACTCGCT GCGCTCGGTC GTTCGGCTGC GGCGAGCGGT 1440 epgfpii ATCAGCTCAC TCAAAGGCGG TAATACGGTT ATCCACAGAA TCAGGGGATA ACGCAGGAAA GAACATGTGA GCAAAAGGCC AGCAAAAGGC 1530 epgfpii CAGGAACCGT AAAAAGGCCG CGTTGCTGGC GTTTTTCCAT AGGCTCCGCC CCCCTGACGA GCATCACAAA AATCGACGCT CAAGTCAGAG 1620 epgfpii GTGGCGAAAC CCGACAGGAC TATAAAGATA CCAGGCGTTT CCCCCTGGAA GCTCCCTCGT GCGCTCTCCT GTTCCGACCC TGCCGCTTAC 1710 epgfpii CGGATACCTG TCCGCCTTTC TCCCTTCGGG AAGCGTGGCG CTTTCTCAAT GCTCACGCTG TAGGTATCTC AGTTCGGTGT AGGTCGTTCG 1800 epgfpii CTCCAAGCTG GGCTGTGTGC ACGAACCCCC CGTTCAGCCC GACCGCTGCG CCTTATCCGG TAACTATCGT CTTGAGTCCA ACCCGGTAAG 1890 epgfpii ACACGACTTA TCGCCACTGG CAGCAGCCAC TGGTAACAGG ATTAGCAGAG CGAGGTATGT AGGCGGTGCT ACAGAGTTCT TGAAGTGGTG 1980 epgfpii GCCTAACTAC GGCTACACTA GAAGGACAGT ATTTGGTATC TGCGCTCTGC TGAAGCCAGT TACCTTCGGA AAAAGAGTTG GTAGCTCTTG 2070 epgfpii ATCCGGCAAA CAAACCACCG CTGGTAGCGG TGGTTTTTTT GTTTGCAAGC AGCAGATTAC GCGCAGAAAA AAAGGATCTC AAGAAGATCC 2160 epgfpii TTTGATCTTT TCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC 2250 epgfpii CTAGATCCTT TTAAATTAAA AATGAAGTTT TAAATCAATC TAAAGTATAT ATGAGTAAAC TTGGTCTGAC AGTTACCAAT GCTTAATCAG 2340 epgfpii TGAGGCACCT ATCTCAGCGA TCTGTCTATT TCGTTCATCC ATAGTTGCCT GACTCCCCGT CGTGTAGATA ACTACGATAC GGGAGGGCTT 2430 epgfpii ACCATCTGGC CCCAGTGCTG CAATGATACC GCGAGACCCA CGCTCACCGG CTCCAGATTT ATCAGCAATA AACCAGCCAG CCGGAAGGGC 2520 epgfpii CGAGCGCAGA AGTGGTCCTG CAACTTTATC CGCCTCCATC CAGTCTATTA ATTGTTGCCG GGAAGCTAGA GTAAGTAGTT CGCCAGTTAA 2610 epgfpii TAGTTTGCGC AACGTTGTTG CCATTGCTAC AGGCATCGTG GTGTCACGCT CGTCGTTTGG TATGGCTTCA TTCAGCTCCG GTTCCCAACG 2700 epgfpii ATCAAGGCGA GTTACATGAT CCCCCATGTT GTGCAAAAAA GCGGTTAGCT CCTTCGGTCC TCCGATCGTT GTCAGAAGTA AGTTGGCCGC 2790 epgfpii AGTGTTATCA CTCATGGTTA TGGCAGCACT GCATAATTCT CTTACTGTCA TGCCATCCGT AAGATGCTTT TCTGTGACTG GTGAGTACTC 2880 epgfpii AACCAAGTCA TTCTGAGAAT AGTGTATGCG GCGACCGAGT TGCTCTTGCC CGGCGTCAAT ACGGGATAAT ACCGCGCCAC ATAGCAGAAC 2970 epgfpii TTTAAAAGTG CTCATCATTG GAAAACGTTC TTCGGGGCGA AAACTCTCAA GGATCTTACC GCTGTTGAGA TCCAGTTCGA TGTAACCCAC 3060 epgfpii TCGTGCACCC AACTGATCTT CAGCATCTTT TACTTTCACC AGCGTTTCTG GGTGAGCAAA AACAGGAAGG CAAAATGCCG CAAAAAAGGG 3150 epgfpii AATAAGGGCG ACACGGAAAT GTTGAATACT CATACTCTTC CTTTTTCAAT ATTATTGAAG CATTTATCAG GGTTATTGTC TCATGAGCGG 3240 epgfpii ATACATATTT GAATGTATTT AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA CCTGACGCGC CCTGTAGCGG 3330 epgfpii CGCATTAAGC GCGGCGGGTG TGGTGGTTAC GCGCAGCGTG ACCGCTACAC TTGCCAGCGC CCTAGCGCCC GCTCCTTTCG CTTTCTTCCC 3420 epgfpii TTCCTTTCTC GCCACGTTCG CCGGCTTTCC CCGTCAAGCT CTAAATCGGG GGCTCCCTTT AGGGTTCCGA TTTAGTGCTT TACGGCACCT 3510 epgfpii CGACCCCAAA AAACTTGATT AGGGTGATGG TTCACGTAGT GGGCCATCGC CCTGATAGAC GGTTTTTCGC CCTTTGACGT TGGAGTCCAC 3600 epgfpii GTTCTTTAAT AGTGGACTCT TGTTCCAAAC TGGAACAACA CTCAACCCTA TCTCGGTCTA TTCTTTTGAT TTATAAGGGA TTTTGCCGAT 3690 epgfpii TTCGGCCTAT TGGTTAAAAA ATGAGCTGAT TTAACAAAAA TTTAACGCGA ATTTTAACAA AATATTAACG TTTACAATTT CCCATTCGCC 3780 epgfpii ATTCAGGCTG CGCAACTGTT GGGAAGGGCG ATCGGTGCGG GCCTCTTCGC TATTACGCCA GCCCAAGCTA CCATGATAAG TAAGTAATAT 3870 epgfpii TAAGGTACGG GAGGTACTTG GAGCGGCCGC AATAAAATAT CTTTATTTTC ATTACATCTG TGTGTTGGTT TTTTGTGTGA ATCGATAGTA 3960 epgfpii CTAACATACG CTCTCCATCA AAACAAAACG AAACAAAACA AACTAGCAAA ATAGGCTGTC CCCAGTGCAA GTGCAGGTGC CAGAACATTT 4050 epgfpii CTCTATCGAT A 4061 epgfpii
Download FASTA file for EpGFPII
REFERENCES
Yuh, C. H., Moore, J. G. and Davidson, E. H. Quantitative functional interrelations within the cis-regulatory system of the S. purpuratus Endo16 gene. Development 122, 4045-4056, 1996.[open]
Yuh, C.-H. and Davidson, E. H. Modular cis-regulatory organization of Endo16, a gut-specific gene of the sea urchin embryo. Development 122, 1069-1082, 1996.[open]
Yuh, C.-H., Bolouri, H. and Davidson, E. H. Genomic cis-regulatory logic: Functional analysis and computational model of a sea urchin gene control system. Science 279, 1896-1902, 1998. [open]
Yuh, C.-H., Bolouri, H. and Davidson, E. H. cis-Regulatory logic in the endo16 gene: Switching from a specification to a differentiation mode of control. Development 128, 617-628, 2001. [open]
Zeller, R. W., Coffman, J. A., Harrington, M. G., Britten, R. J. and Davidson, E. H. SpGCF1, a sea urchin embryo transcription factor, exists as five nested variants encoded by a single mRNA. Dev. Biol. 169, 713-727, 1995. [open]
Arnone, M. and Davidson, E. H. The hardwiring of development: Organization and function of genomic regulatory systems. Development 124, 1851-1864, 1997.
Arnone, M. I., Martin, E. L. and Davidson. E. H. Cis-regulation downstream of cell type specification: A single compact element controls the complex expression of the CyIIa gene in sea urchin embryos. Development 125, 1381-1395, 1998. [open]