BBa_J18951
1
BBa_J18951
FRB ~ mCitrine
2010-01-26T12:00:00Z
2015-08-31T04:08:36Z
synthetic
P19 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
component2066800
1
BBa_B0105
component2066794
1
BBa_J18919
component2066790
1
BBa_B0105
component2066782
1
BBa_J18912
component2066802
1
BBa_J18913
component2066788
1
BBa_J18922
component2066791
1
BBa_J18931
component2066785
1
BBa_J18926
component2066798
1
BBa_J18909
component2066793
1
BBa_B0105
component2066784
1
BBa_B0105
component2066787
1
BBa_B0105
component2066796
1
BBa_B0105
annotation2066796
1
BBa_B0105
range2066796
1
1155
1160
annotation2066798
1
BBa_J18909
range2066798
1
1161
1178
annotation2066800
1
BBa_B0105
range2066800
1
1179
1184
annotation2066793
1
BBa_B0105
range2066793
1
1125
1130
annotation2066784
1
BBa_B0105
range2066784
1
84
89
annotation2066785
1
BBa_J18926
range2066785
1
90
368
annotation2066791
1
BBa_J18931
range2066791
1
411
1124
annotation2066802
1
BBa_J18913
range2066802
1
1185
1319
annotation2066787
1
BBa_B0105
range2066787
1
369
374
annotation2066782
1
BBa_J18912
range2066782
1
1
83
annotation2066794
1
BBa_J18919
range2066794
1
1131
1154
annotation2066788
1
BBa_J18922
range2066788
1
375
404
annotation2066790
1
BBa_B0105
range2066790
1
405
410
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_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_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_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_J18931
1
mCitrine
mCitrine
2010-01-26T12:00:00Z
2015-08-31T04:08:36Z
gene synthesis
Yellow fluorescent protein mCitrine.
===See also===
* BBa_J18930
===References===
* Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY. (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. PMID: 11387331
false
false
_165_
0
2175
165
It's complicated
false
* starts with consensus VSKGEEL
* ends with consensus DELYK
false
Raik Gruenberg
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_J18922_sequence
1
ggtagcggcagcggtagcggtagcggcagc
BBa_B0105_sequence
1
accggc
BBa_J18951_sequence
1
taatacgactcactatagggagaccacaacggtttccctctagcaataattttgtttaactttaagaaggagatatacatatgaccggcatcctgtggcatgaaatgtggcatgaaggcctggaagaagcgagccgtctgtattttggcgaacgtaacgtgaaaggcatgtttgaagtgctggaaccgctgcatgccatgatggaacgtggcccgcagaccctgaaagaaaccagctttaaccaggcgtatggccgtgatctgatggaagcgcaggaatggtgccgtaaatacatgaaaagcggcaacgtgaaagatctgctgcaagcgtgggatctgtattatcatgtgtttcgccgcatcagcaaaaccggcggtagcggcagcggtagcggtagcggcagcaccggcgtgagcaaaggcgaagaactgtttaccggcgtggtgccgattctggtggaactggatggtgatgtgaacggccataaatttagcgtgagcggcgaaggcgaaggtgatgcgacctatggcaaactgaccctgaaatttatttgcaccacgggtaaactgccggttccgtggccgaccctggtgaccacctttggctatggcctgatgtgctttgcgcgttatccggatcatatgaaacagcacgatttctttaaaagcgccatgccggaaggctatgtgcaggaacgcaccatcttttttaaagatgatggcaactataaaacccgtgcggaagtgaaatttgaaggcgataccctggtgaaccgtattgaactgaaaggcatcgatttcaaagaagatggcaacattctgggccataaactggaatataactacaacagccataacgtgtatatcatggcggataaacagaaaaacggcatcaaagtgaactttaaaatccgccacaacattgaagatggcagcgtgcagctggccgatcattatcagcagaacaccccgattggtgatggcccggtgctgctgccggataaccattatctgagctaccagagcaaactgagcaaagatccgaacgaaaaacgtgatcacatggtgctgctggaatttgtgaccgcagccggtattaccctgggcatggatgaactgtacaaaaccggcctggaagtgctgtttcagggcccgaccggccatcatcatcatcatcataccggctagtgactgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggat
BBa_J18913_sequence
1
tagtgactgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaaggaggaactatatccggat
BBa_J18909_sequence
1
catcatcatcatcatcat
BBa_J18912_sequence
1
taatacgactcactatagggagaccacaacggtttccctctagcaataattttgtttaactttaagaaggagatatacatatg
BBa_J18926_sequence
1
atcctgtggcatgaaatgtggcatgaaggcctggaagaagcgagccgtctgtattttggcgaacgtaacgtgaaaggcatgtttgaagtgctggaaccgctgcatgccatgatggaacgtggcccgcagaccctgaaagaaaccagctttaaccaggcgtatggccgtgatctgatggaagcgcaggaatggtgccgtaaatacatgaaaagcggcaacgtgaaagatctgctgcaagcgtgggatctgtattatcatgtgtttcgccgcatcagcaaa
BBa_J18931_sequence
1
gtgagcaaaggcgaagaactgtttaccggcgtggtgccgattctggtggaactggatggtgatgtgaacggccataaatttagcgtgagcggcgaaggcgaaggtgatgcgacctatggcaaactgaccctgaaatttatttgcaccacgggtaaactgccggttccgtggccgaccctggtgaccacctttggctatggcctgatgtgctttgcgcgttatccggatcatatgaaacagcacgatttctttaaaagcgccatgccggaaggctatgtgcaggaacgcaccatcttttttaaagatgatggcaactataaaacccgtgcggaagtgaaatttgaaggcgataccctggtgaaccgtattgaactgaaaggcatcgatttcaaagaagatggcaacattctgggccataaactggaatataactacaacagccataacgtgtatatcatggcggataaacagaaaaacggcatcaaagtgaactttaaaatccgccacaacattgaagatggcagcgtgcagctggccgatcattatcagcagaacaccccgattggtgatggcccggtgctgctgccggataaccattatctgagctaccagagcaaactgagcaaagatccgaacgaaaaacgtgatcacatggtgctgctggaatttgtgaccgcagccggtattaccctgggcatggatgaactgtacaaa
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