BBa_J18912 1 T7start T7 promoter + RBS + START 2010-01-26T12:00:00Z 2015-08-31T04:08:36Z pET3a expression vector Strong T7 promoter and RBS including start codon for protein expression in E. coli or in-vitro. Part is provided in RFC 25 assembly format. false false _165_ 0 2175 165 It's complicated false * sequence is taken from the commonly used pET3a expression vector * deleted XbaI site between prom. and RBS * T7 promoter sequence (1-19) equals the one in BBa_I712074 * Conservative choice of part boundaries and modifications -- the part's size could probably be reduced. false Raik Gruenberg annotation2065586 1 start range2065586 1 81 83 BBa_J18926 1 FRB FRB(T2098L) 2010-01-26T12:00:00Z 2015-08-31T04:08:36Z human mTOR (aa sequence) gene synthesis Modified FKBP12-rapamycin-binding domain (FRB) of human mTOR Kinase (=mammalian Target of Rapamycin, also called FRAP = FKBP12-rapamycin associated protein). In presence of the immunosupressant drug rapamycin, FRB forms a high-affinity complex with FKBP12. There is no detectable FKBP12 - FRB interaction in absence of rapamycin. ===Modification for a non-toxic dimerizer=== Rapamycin arrests growth and proliferation of many cell types (reviewed in Jacinto and Hall, 2003) by inhibiting protein translation. The mutation T2089L (mTor numbering) makes FRB binding to a non-toxic Rapamycin analogue (rapalogue) AP21967 as well as the original Rapamycin. The FRB - FKBP12 heterodimerization can then be triggered by both Rapamycin and the non-toxic rapalogue. This variant of FRB and the synthetic dimerizer AP21967 are the basis of the Argent(tm) heterodimerization kit from Ariad Pharmaceuticals Inc. '''Note:''' Two additional mutations would convert FRB into FRB* which is used for conditional (drug-reversible) degradation of fusion proteins. These two additional mutations are not implemented in this part. ===Protein Parameters:=== * Number of amino acids: 93 * Molecular weight: 11285.9 * Theoretical pI: 6.47 false false _165_ 0 2175 165 It's complicated false * aa translation from human mTOR, * mutation T2089L (verified against pC4-RHE from Ariad) * GeneArt codon optimization for E. coli * gene synthesis false Raik Gruenberg BBa_J18952 1 BBa_J18952 FRB ~ mCerulean 2010-01-26T12:00:00Z 2015-08-31T04:08:36Z synthetic P20 expression cassette from Gruenberg et al. 2010 series of interaction reporter proteins. All proteins share the same architecture: * T7Start-domain1-5xGS-domain2-preSCsite-HisTag-T7Stop Only Domains1 and 2 vary and are taken from a list of protein interaction input and output (readout) parts. ===Expression=== Proteins were expressed in E. coli BL21 using T7 Polymerase and were purified by His-tag affinity. ===Reference=== * Gruenberg, Ferrar, van der Sloot, Serrano (2010): Building Blocks for Protein Interaction Devices false false _165_ 0 2175 165 It's complicated false Constructs were assembled according to R.F.C. 25. All parts were codon optimized for E. coli. false Raik Gruenberg component2066818 1 BBa_B0105 component2066807 1 BBa_J18926 component2066822 1 BBa_B0105 component2066820 1 BBa_J18909 component2066813 1 BBa_J18930 component2066815 1 BBa_B0105 component2066809 1 BBa_B0105 component2066810 1 BBa_J18922 component2066806 1 BBa_B0105 component2066816 1 BBa_J18919 component2066804 1 BBa_J18912 component2066824 1 BBa_J18913 component2066812 1 BBa_B0105 annotation2066809 1 BBa_B0105 range2066809 1 369 374 annotation2066818 1 BBa_B0105 range2066818 1 1155 1160 annotation2066815 1 BBa_B0105 range2066815 1 1125 1130 annotation2066816 1 BBa_J18919 range2066816 1 1131 1154 annotation2066824 1 BBa_J18913 range2066824 1 1185 1319 annotation2066806 1 BBa_B0105 range2066806 1 84 89 annotation2066822 1 BBa_B0105 range2066822 1 1179 1184 annotation2066807 1 BBa_J18926 range2066807 1 90 368 annotation2066804 1 BBa_J18912 range2066804 1 1 83 annotation2066812 1 BBa_B0105 range2066812 1 405 410 annotation2066813 1 BBa_J18930 range2066813 1 411 1124 annotation2066810 1 BBa_J18922 range2066810 1 375 404 annotation2066820 1 BBa_J18909 range2066820 1 1161 1178 BBa_J18930 1 mCerulean mCerulean CFP 2010-01-26T12:00:00Z 2015-08-31T04:08:36Z literature gene synthesis Cerulean GFP is an optimized FRET donor molecule that was rationally designed by Piston and co-workers to eliminate the excited-state heterogeneity of ECFP. In ECFP, a single-exponential fit to the fluorescence lifetime is not feasible due to multiple components, whereas Cerulean exhibits essentially homogeneous excited-state decay kinetics, rendering this protein useful for lifetime imaging... Live cell imaging experiments have demonstrated that Cerulean GFP undergoes highly efficient FRET to yellow acceptor molecules (25, 26). Additionally, Cerulean exhibits more than twice the brightness of ECFP and CyPet, emitting light at fluorescence intensities similar to Citrine (20). ===References=== * Rizzo MA, Springer GH, Granada B, Piston DW. (2004) An improved cyan fluorescent protein variant useful for FRET. PMID: 14990965 false false _165_ 0 2175 165 It's complicated false * amino acid sequence taken from a pAG304GAL-Cerulean-ccdB from LindQuist lab * borders trimmed to match the construct used for PDB 2Q57 (last 9 AA are not resolved in PDB) * structure starts with MSK, this construct starts with MVSK but the M has been skipped for this Biobrick * gene synthesis * codon optimized for E. coli false Raik Gruenberg BBa_J18909 1 HisTag His tag (6xHis; E. coli - optimized) 2010-01-15T12:00:00Z 2015-08-31T04:08:35Z Synthetic DNA Hexahistidine affinity tag. Binds zinc, nickel, or cobalt affinity resins. =Notes= This part is identical to BBa_I757013 (which does not seem to be available though). =References= Wikipedia article: [http://en.wikipedia.org/wiki/Polyhistidine-tag] false false _165_ 0 2175 165 It's complicated false Simple repetition of the His codon most frequent in E. coli -- a more balanced codon-choice may be better. false Raik Gruenberg annotation2065463 1 PolyHis range2065463 1 1 18 BBa_J18922 1 5xGS 10aa [GS]x linker 2010-01-26T12:00:00Z 2015-08-31T04:08:36Z gene synthesis Highly flexible 10 amino acid linker. Translates to [GS]_5. Codon-optimize for E. coli, yeast, mammalian. false true _165_ 0 2175 165 It's complicated false * gene synthesis * codon optimized for E. coli * experimental distance distributions are described in Neuweiler et al. 2006 papers. false Raik Gruenberg BBa_J18913 1 T7stop T7 terminator (incl. STOP) 2010-01-26T12:00:00Z 2015-08-31T04:08:36Z pET3a expression plasmid T7 terminator for protein expression in E. coli or in vitro. Part is provided in R.F.C. 25 assembly format. false false _165_ 0 2175 165 It's complicated false * taken from pET3a expression plasmid * added two STOP to 5' * conservative choice of part boundary, could probably be shortened false Raik Gruenberg annotation2065587 1 stop range2065587 1 1 6 BBa_B0105 1 Scar 25 RFC 25 Scar Sequence 2009-10-14T11:00:00Z 2015-08-31T04:07:21Z BBF RFC 25 This is the scar produced by assembly using RFC 25. If you are assembling a composite part using RFC 25, you can insert this part and specify blunt assembly to get the desired sequence. false false _1_ 0 25 397 Not in stock false Simple DNA sequence false Randy Rettberg annotation2041621 1 Scar 25 range2041621 1 1 6 BBa_J18919 1 preSCsite preScission protease cleavage site (E. coli-optimized) 2010-01-26T12:00:00Z 2015-08-31T04:08:36Z gene synthesis cleavage site for PreScission??? protease. preScission protease cleavage site (PreScission??? protease is a genetically engineered form of human rhinovirus 3C protease with a GST fusion, allowing for facile cleavage and purification of GST-tagged proteins along with protease removal after recombinant protein cleavage. Enables low-temperature cleavage of fusion proteins containing the eight-residue recognition sequence.) cleavage site: * Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro References ============== Walker et al. (1994): Efficient and rapid affinity purification of proteins using recombinant fusion proteases. PMID: 7764949 false false _165_ 0 2175 165 It's complicated false * gene synthesis * codon optimized for E. coli false Raik Gruenberg BBa_J18952_sequence 1 taatacgactcactatagggagaccacaacggtttccctctagcaataattttgtttaactttaagaaggagatatacatatgaccggcatcctgtggcatgaaatgtggcatgaaggcctggaagaagcgagccgtctgtattttggcgaacgtaacgtgaaaggcatgtttgaagtgctggaaccgctgcatgccatgatggaacgtggcccgcagaccctgaaagaaaccagctttaaccaggcgtatggccgtgatctgatggaagcgcaggaatggtgccgtaaatacatgaaaagcggcaacgtgaaagatctgctgcaagcgtgggatctgtattatcatgtgtttcgccgcatcagcaaaaccggcggtagcggcagcggtagcggtagcggcagcaccggcgtgagcaaaggcgaagaactgtttaccggcgtggtgccgattctggtggaactggatggtgatgtgaacggccataaatttagcgtgagcggcgaaggcgaaggtgatgcgacctatggcaaactgaccctgaaattcatttgcaccaccggcaaactgccggttccgtggccgaccctggttaccaccctgacctggggcgtgcagtgctttgcgcgttatccggatcatatgaaacagcacgatttctttaaaagcgccatgccggaaggctatgtgcaggaacgcaccatcttttttaaagatgatggcaactataaaacccgtgcggaagtgaaatttgaaggcgataccctggtgaaccgtattgaactgaaaggcatcgatttcaaagaagatggcaacattctgggccataaactggaatataactacatcagcgataacgtgtatatcaccgcggataaacagaaaaacggcatcaaagcgaactttaaaatccgccacaacattgaagatggcagcgtgcagctggccgatcattatcagcagaacaccccgattggtgatggcccggtgctgctgccggataaccattatctgagcacccagagcaaactgagcaaagatccgaacgaaaaacgtgatcacatggtgctgctggaatttgtgaccgcagccggtattaccctgggcatggatgaactgtacaaaaccggcctggaagtgctgtttcagggcccgaccggccatcatcatcatcatcataccggctagtgactgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggat BBa_J18922_sequence 1 ggtagcggcagcggtagcggtagcggcagc BBa_B0105_sequence 1 accggc BBa_J18913_sequence 1 tagtgactgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggat BBa_J18909_sequence 1 catcatcatcatcatcat BBa_J18930_sequence 1 gtgagcaaaggcgaagaactgtttaccggcgtggtgccgattctggtggaactggatggtgatgtgaacggccataaatttagcgtgagcggcgaaggcgaaggtgatgcgacctatggcaaactgaccctgaaattcatttgcaccaccggcaaactgccggttccgtggccgaccctggttaccaccctgacctggggcgtgcagtgctttgcgcgttatccggatcatatgaaacagcacgatttctttaaaagcgccatgccggaaggctatgtgcaggaacgcaccatcttttttaaagatgatggcaactataaaacccgtgcggaagtgaaatttgaaggcgataccctggtgaaccgtattgaactgaaaggcatcgatttcaaagaagatggcaacattctgggccataaactggaatataactacatcagcgataacgtgtatatcaccgcggataaacagaaaaacggcatcaaagcgaactttaaaatccgccacaacattgaagatggcagcgtgcagctggccgatcattatcagcagaacaccccgattggtgatggcccggtgctgctgccggataaccattatctgagcacccagagcaaactgagcaaagatccgaacgaaaaacgtgatcacatggtgctgctggaatttgtgaccgcagccggtattaccctgggcatggatgaactgtacaaa BBa_J18912_sequence 1 taatacgactcactatagggagaccacaacggtttccctctagcaataattttgtttaactttaagaaggagatatacatatg BBa_J18926_sequence 1 atcctgtggcatgaaatgtggcatgaaggcctggaagaagcgagccgtctgtattttggcgaacgtaacgtgaaaggcatgtttgaagtgctggaaccgctgcatgccatgatggaacgtggcccgcagaccctgaaagaaaccagctttaaccaggcgtatggccgtgatctgatggaagcgcaggaatggtgccgtaaatacatgaaaagcggcaacgtgaaagatctgctgcaagcgtgggatctgtattatcatgtgtttcgccgcatcagcaaa BBa_J18919_sequence 1 ctggaagtgctgtttcagggcccg igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 James Alastair McLaughlin Chris J. Myers 2017-03-06T15:00:00.000Z