News & Highlights: A recent collabaration between YRC researcher John Yates and Martin W. Hetzer applied proteomics to examine protein turnover in cells of the rat central nervous system. They found that extremely long-lived proteins associated with chromatin and the nuclear pore complex did not turn over, potentially exposing these proteins to harmful metabolites and accumulation of damage over time. Read more about their findings in the journal Science to learn more. [Read Article]
News & Highlights: YRC Researchers Michael MacCoss and William Stafford Noble have published a new algorithm, dubbed Barista, for identifying proteins in complex biological mixtures. Instead of subdividing the task into separate peptide and protein identification tasks, Barista applies a machine learning approach to identify proteins from source spectra as a single optimization problem. Read their publications in Mol. Cell Proteomics to learn more. [Read Article]
News & Highlights: YRC researcher Stan Fields has used protein mass spectrometry to identify 870 unique sites of ubiquitin attachment on 438 different proteins in the budding yeast Saccharomyces cerevisiae. The analysis was based on the increase in molecular mass of a tryptic peptide carrying two additional glycine residues from the ubiquitin moiety. Read his paper in Proteomics to learn more. [Read Article]
News & Highlights: Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal disease characterized by premature aging. In their recent collaboration, YRC researcher John Yates and collaborator Juan Carlos Izpisua Belmonte found induced pluripotent stem cells from HGPS patients lacked molecular characteristics associated with the disease, which were restored upon differentiation. See their paper in Nature to learn more. [Read Article]
News & Highlights: YRC researcher Stan Fields used the model organism Saccharomyces cerevisiae to probe the effects of nutritionally acquired metabolites on statins, a cholesterol-lowering drug widely prescribed to prevent heart disease. He found that copper and zinc ions impair the effect of statins by upregulating genes related to sterol production. Please read his paper in Molecular BioSystems to learn more. [Read Article]
News & Highlights: YRC researchers David Baker and Stan Fields have developed new technology for examining how a protein's sequence affects its function. This new technology is large-scale and may be applied to many in vitro or in vivo protein assays, providing a general means for studying the functional consequences of protein variation. Please read their paper in Nature Methods to learn more. [Read Article]
News & Highlights: The YRC collaborated with Sue Biggins at the Fred Hutchinson Cancer Research Center in Seattle to examine centromeres, whose proper function is critical to prevent conditions associated with cancer and some birth defects. This work, performed in yeast, was recently published in Molecular Cell, where Dr. Biggins proposes a new pathway for the regulation of centromeric function. [Read Article]
News & Highlights: Multidimensional protein identification technology (MudPIT) developed by the YRC was used in a recent collaboration with David Drubin at the University of California, Berkeley, to examine the assembly of actin networks in yeast. In his recent paper in Current Biology, Dr. Drubin describes the nucleation and assembly of these large protein complexes, and how MudPIT was used to characterize their composition. [Read Article]

CFP (kan) [plasmid pDH3]

Plamid pDH3 contains the ORF for the cyan-fluorescent protein (CFP), a variant of GFP with a blue-shifted spectrum. The plasmid also contains the Kan selectable marker encoding resistance to the antibiotic G418. The codons have not been optimized for mammalian expression and instead are the same as in the parent vector (except for the 6 mutations listed below). Forward and reverse primers refer to the sequence of the primers described on the protocol page "Fusing GFP to the Carboxy Terminus of Your Favorite Protein." Some common restriction enzyme sites are shown. Unique sites are in red.

Map

Parent Vector

pFA6a-GFP(S65T)-Kan. See Wach et al.

Mutations

Amino Acid Change Original Codon New Codon
F64L TTC TTG
Y66W TAT TGG
N146I AAC ATT
M153T ATG ACT
V163A GTT GCT
N164H AAT CAT
Also includes S65T and Q80R from parent vector. Q80R is believed to be a neutral mutation.

Sequence

The DNA sequence of pDH3 is shown below with the CFP sequence in blue.
GAACGCGGCCGCCAGCTGAAGCTTCGTACGCTGCAGGTCGACGGATCCCC GGGTTAATTAACAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAAT TCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTG GAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATT TGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTT GACTTGGGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAACGGC ATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACT ATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTT TGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTA AAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATATTTCA CACAATGTATACATCACTGCAGACAAACAAAAGAATGGAATCAAAGCTCA TTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACC ATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGAC AACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAA GAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACAC ATGGCATGGATGAACTATACAAATAGGGCGCGCCACTTCTAAATAAGCGA ATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATA AGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTT ATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGT ATGAGGTCGCTCTTATTGACCACACCTCTACCGGCAGATCCGCTAGGGAT AACAGGGTAATATAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACC CGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTG CGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACA TCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGC GAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCT CCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAA TATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTT TTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAA CCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAAC ATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCA ATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGT TGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAG ATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAA GCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCC CCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAA AATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCC TGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGG CGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGAC GAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCT TTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTG ATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGA CGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTG CCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATG GTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGAT GAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAAC TTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAA TGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATG CGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATG CTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTTTA AACGAGCTCGAATTCATCGATGATATCAGATCCACTAGTGGCCTATGCGG CCGCGGATCTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGC CAGGTTAACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTT GCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGG TCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGG TTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGG CCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGT GCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATC GTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTA TCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCT TGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAA GCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCT TTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT TTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTA AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTG ACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTA TTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGAT ACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACC CACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGG GCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTAT TAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTT GGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATG ATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCG TTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA GTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGA ACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC AAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCAC CCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCA AAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAA ATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAAT AAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGT CTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCA CGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGA CACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGG GAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGG GCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCAT ATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATG TATCATACACATACGATTTAGGTGACACTATA

Plasmids & Protocols