Libprepd and Epgfp: Difference between pages

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imported>Echinobase
(Created page with " <p>NB: These are additional notes, mostly from Carolina Livi, that supplement the Gibco-BRL Superscript cDNA library kit manual. The pla...")
 
imported>Ctelmer
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<p>NB: These are additional notes, mostly from Carolina Livi, that supplement the Gibco-BRL Superscript cDNA library kit manual.  The plasmid vector is called pSPORT.  This description specifically pertains to the embryonic libraries prepared in the Davidson laboratory (7hr, 15hr, 20hr, 30hr, 40hr). The other cDNA libraries are all in pSport [[http://www.spbase.org/SpBase/resources/methods/cDNAvector.txt ''Sequence'']] but the details of preparation may differ in the Livingston and Ettensohn laboratories.</p>
<h3>Kits</h3>
<ul><p><b>Total RNA</b>. we use RNAzol TM B from Leedo Medical Laboratories Catalog CS-104 for 100 ml or Catalog CS-105 for 200ml. We follow the protocol attached with it.</p>
<p><b>mRNA Isolation</b>. To isolate mRNA you will need Dynabeads Oligo (dT)25 from Dynal(phone  800 638 9416) Product No. 610.02 for 2 ml or 610.05 for 5 ml.</p>
<p><b>Library</b>. he kit we use to make the library is SuperScript TM Plasmid System for cDNA Synthesis and Plasmid Cloning from GibcoBRL Catalog  18248-013 (you can make 3 libraries with the kits, but in my experience you run out of tRNA, which can be purchases separately or you can use some you have hanging around). Instead of using the column from the Gibco kit, we used one provided by cDNA Synthesis Kit from Stratagene Catalog  200401.</p>
</ul><h3>Procedures</h3>
<ul><p><b>RNA Isolation</b>. Here is the procedure we use to isolate mRNA with the Dynabeads. Basically we have combined many of the protocols suggested on their information sheet.</p>
</ul><ul><p>mRNA isolation with Dynabead (oligo dT) from total RNA </p>
<p>1) Dispense 600ul of beads, wash with 2X BINDING BUFFER.</p>
<p>2) add 300 ul 2X BINDING BUFFER + 300ul (225ug) total RNA (0.75ug/ul).</p>
<p>3) wash 3X with 600ul 1X WASHING BUFFER.</p>
<p>4) elute with 60ul of RNase free water.</p>
<p>5) wash the beads with 600ul of 1X WASHING BUFFER 2 times.</p>
<p>6) wash beads with 300 ul of 2X BINDING BUFFER </p>
<p>7) repeat steps 2-6,4 times.</p>
<p>8) precipitate mRNA  add to the 300 ul mRNA in ddH2O obtained above:</p>
<p>30ul 3M NaOAc</p>
<p>660ul 100% EtOH</p>
<p>keep at -20oC O/N or until needed</p>
<p>Approximate yield: 30ug mRNA / 1000ug total RNA Before using mRNA from library, run ~ 1ug in agarose gel (boil the mRNA for no longer than 3 min). The recipe for the BINDING and WASHING BUFFERS is in the Dynabeads information sheet.</p>
<h3>Amounts </h3>
<p>For the cDNA library one should start with 5 ug of mRNA.  We followed the standard protocol from the kit except we used our own primers (see below) not those provided with the kit. We add the SuperScript II enzyme at room temperature instead of at 37°C. We get between 5X 10E5 - 5 X 10E6 clones when we start with 5 ug of mRNA.</p>
<h3>Bacteria</h3>
<p>It's the ElectroMAX DH10B TM cells from GibcoBRL Catalog 18290-015 (size 0.5ml). This item is often backordered and should be ordered in advance. The bacteria should not be stored for a long time (more than 3 months) for the purpose of the libraries(although leftovers can be used for other applications). The 500 ul will probably be enough for 1 library, but if you have other uses for them I would have 1 ml available.  We follow the transformation procedure provided in the information sheet.</p>
<h3>Storage of libraries</h3>
<p>We usually freeze (-70) the transformed bacteria (after plating 10ul of a 1 ml aliquot to check the transformation efficiency and colony density. We tend to get at least 90% of the colony density after thawing, so we don't find this a minus.</p>
<h3>Primers</h3>
<p>Below is the description of the primers we used to make our libraries.</p>
<p></p>
<p>A A A G G A A G G A A A A A A G C G G C C G C T A C A N(8) T  </p>
<p>______________________________----------------_______ ------</p>
</ul><pre>            XO Block          Not I site  marker  random</pre><p>
<br></p>
<span style="font-size:92%; line-height: 2em;">The sequence marked XO Block protects the Not I site against nuclease activity. The TACA of the marker sequence helps todifferentiate the primer from naturally occurring Not I sites. The 8 random nucleotides are next.  The trailing T is an artifactof the old chemistry when a specific nucleotide was used on the synthesis column. Newer chemistry allows any nucleotide in that position.


It is important to gel purify this oligo, for a truncated form containing the random sequence but not the Not I site would impair the cloning of the cDNA fragments.
<p>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 [http://www.fpbase.org/protein/gfp-s65t/ 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.</p>
</span>
 
 
== EPGFP2 ==
 
[[image:Small_map.jpg||left|700x700px|EpGFPII plasmid vector]]
 
 
<div class="toccolours mw-collapsible mw-collapsed" style="clear:both">
EpGFPII FASTA Sequence
<div class="mw-collapsible-content">>epgfpii
<pre>
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
</pre></div>
</div>
[http://ftp.echinobase.org/pub/WikiDownloads/epgfpii.fasta 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.<span class="newwin">[https://new.echinobase.org/literature/article.do?method=display&articleId=36543  [open<nowiki>]</nowiki>]</span>
 
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.<span class="newwin">[http://new.echinobase.org/literature/article.do?method=display&articleId=36421  [open<nowiki>]</nowiki>]</span>
 
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.
<span class="newwin">[http://new.echinobase.org/literature/article.do?method=display&articleId=36860  [open<nowiki>]</nowiki>]</span>
 
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.
<span class="newwin">[http://new.echinobase.org/literature/article.do?method=display&articleId=37679  [open<nowiki>]</nowiki>]</span>
 
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.
<span class="newwin">[http://new.echinobase.org/literature/article.do?method=display&articleId=35795  [open<nowiki>]</nowiki>]</span>
 
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.
<span class="newwin">[http://new.echinobase.org/literature/article.do?method=display&articleId=36858  [open<nowiki>]</nowiki>]</span>

Revision as of 15:57, 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.


EPGFP2

EpGFPII plasmid vector
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]

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