Epgfp

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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 plasmid vector


EpGFPII FASTA Sequence

>epgfpii
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]