Team:Freiburg/Parts

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All of our parts feature our expanded Pre- and Suffix for in frame cloning of fusion proteins [http://parts.mit.edu/igem07/index.php/Freiburg07/report_fusion_parts (more)]. A complete, detailed list of all parts submitted to the registry in this year´s project can be found [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Freiburg here], as well as the respective DNA-Sequences.

basic parts

• Signal peptide
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157001 BBa_K157001]
Description:
This sequence mediates transportation of the protein to a translocational pore by an RNA–multiprotein complex, the signal recognition particle (SRP) when fused to the n-terminus of a fusion protein with a transmembrane region[1]. There, after a pause of translation, the signal sequence is released and translation and translocation of the nascent chain are restarted[2].
Source:
Human EGFR (ErbB-1) signal sequence; originally mediating membrane integration of the EGF-receptor (ErbB-1). Sequence taken from UniProtKB/Swiss-Prot entry P00533.
Gene synthesis by ATG:biosynthetics, optimized for expression in homo sapiens by iGEM-Team Freiburg 2008.
References:
[1] Walter P, Johnson AE: “Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane.” Annu Rev Cell Biol 1994, 10:87-119
[2] Robert M Stroud, Peter Walter: “Signal sequence recognition and protein targeting”, Current Opinion in Structural Biology 1999, 9:754–759
AA sequence:
MRPSGTAGAA LLALLAALCP ASRA
gene sequence:
ATGAGACCAT CTGGTACTGC TGGAGCCGCA TTGCTGGCAC TTTTGGCTGC GCTGTGCCCT GCAAGCAGAG CA

• Erbb1-transmembrane region
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157002 BBa_K157002]
Description:
Helical, single-span transmembrane region of the human EGF-Receptor type ErbB-1, sequence taken from UniProtKB/Swiss-Prot entry P00533.
source:
Transmembrane region of the human EGF-Receptor type ErbB-1. Sequence taken from UniProtKB/Swiss-Prot entry P00533. Gene synthesis by ATG:biosynthetics, optimized for expression in homo sapiens by iGEM-Team Freiburg 2008.
AA sequence:
ATGMVGALLL LLVVALGIGL FM
gene sequence:
ATAGCTACCG GAATGGTGGG TGCACTTTTG CTCCTTTTGG TCGTTGCCCT GGGGATAGGA CTCTTTATG

• Sc-Fv anti NIP
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157003 BBa_K157003]
Description:
Singlechain Fv.Fragment of the Anti-Nitro-Iodo-Phenol-antibody B1-8. Binds the hapten NIP (Nitro-Iodo-Phenole) with an affinity of 2.0 uM[1,2]. Designed for fusion to proteins or peptides via in frame cloning.
source:
Gene synthesis by GeneArt, optimized for expression in homo sapiens.
References:
[1]Ana Cumano and Klaus Rajewski: “Clonal recruitment and somatic mutation in the generation of immunological memory to the hapten NP”, The EMBO Journal vol. 5 no.10 pp. 2459-2468, 1986
[2]D. Allen, T. Simon, F. Sablitzky, K. Rajewski and A. Cumano: “Antibody engineering for the analysis of affinity maturation of an anti-hapten response”, The EMBO Journal vol. 7 no.7 pp. 1995-2001, 1988
AASequence:
QVQLQQPGAE LVKPGASVKL SCKASGYTFT SYWMHWVKQR PGRGLEWIGR IDPNSGGTKY NEKFKSKATL TVDKPSSTAY MQLSSLTSED SAVYYCARYD YYGGSYFDYW GQGTTVTVSS GGGGSGGGGS GGGGSQAVVT QESALTTSPG ETVTLTCRSS TGAVTTSNYA NWVQEKPDHL FTGLIGGTNN RAPGVPARFS GSLIGDKAAL TITGAQTEDE AIYFCALWYS NHWVFGGGTK LTVLG
gene sequence:
CAGGTGCAGC TCCAGCAGCC CGGAGCCGAA CTGGTGAAGC CAGGCGCCAG CGTGAAGCTG TCCTGCAAGG CCAGCGGCTA CACCTTCACC AGCTACTGGA TGCACTGGGT GAAACAGAGG CCCGGCAGAG GCCTGGAATG GATCGGCCGG ATCGACCCCA ACAGCGGCGG CACCAAGTAC AACGAGAAGT TCAAGAGCAA GGCCACCCTG ACCGTGGACA AGCCCAGCAG CACCGCCTAC ATGCAGCTGT CCAGCCTGAC CAGCGAGGAC AGCGCCGTGT ACTACTGCGC CAGATACGAC TACTACGGCG GCAGCTACTT CGACTACTGG GGCCAGGGCA CCACCGTGAC CGTGTCCTCT GGGGGAGGGG GCTCAGGAGG AGGAGGAAGC GGGGGAGGGG GCAGCCAGGC CGTGGTGACC CAGGAAAGCG CCCTGACCAC TCCCCTGGCG GACAGTGACC TGACCTGCCG TCCTCTACAG GCGCCGTGAC CACAAGCAAC TACGCCAACT GGGTGCAGGA AAAGCCCGAC CACCTGTTCA CCGGCCTGAT CGGCGGCACA AACAACAGAG CCCCTGGCGT GCCCGCTAGA TTCAGCGGCA GCCTGATCGG GGACAAGGCC GCCCTGACAA TCACAGGCGC CCAGACCGAG GACGAGGCCA TCTACTTTTG CGCCCTGTGG TACAGCAACC ACTGGGTGTT CGGCGGAGGG ACCAAGCTGA CCGTGCTGGG C

• LipocalinFluA
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157004 BBa_K157004]
Description:
Fluoresceine A -binding derivative of a Lipocalin (BBP, bilin binding protein from pieris brassicae), originally designed by Arne Skerra[1,2].
Source:
Gene synthesis by GeneArt, optimized for expression in homo sapiens.
References:
[1] Gerald Beste, Frank S. Schmidt, Thomas Stibora and A. Skerra: “Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold“,Proc. Natl. Acad. Sci. USA Vol. 96, pp. 1898–1903, March 1999 Biochemistry
[2]Ingo P. Korndörfer, Gerald Beste and A. Skerra: “Crystallographic Analysis of an “Anticalin” With Tailored Specificity for Fluorescein Reveals High Structural Plasticity of the Lipocalin Loop Region”, PROTEINS: Structure, Function, and Bioinformatics 53:121–129 (2003)
AA sequence:
MAGDVYHDGA CPEVKPVDNF DWSQYHGKWW EVAKYPSPNG KYGKCGWAEY TPEGKSVKVS RYDVIHGKEY FMEGTAYPVG DSKIGKIYHS RTVGGYTRKT VFNVLSTDNK NYIIGYSCRY DEDKKGHWDH VWVLSRSMVL TGEAKTAVEN YLIGSPVVDS QKLVYSDFSE AACKVNNTG

• Split-Cerulean-C-CFP
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157005 BBa_K157005]
Description:
C-terminal fragment (Amino acids156-236) of the monomeric[5] cyan fluorescent protein variant “Cerulean”, designed for fusion/BiFC[1,3]. Reassembly with Split-Cerulean-nCFP (Part Bba_K157006) generates the complete, working CFP “Cerulean”, whereas combination with the N-terminal fragment (Part Bba_K157008) of the monomeric yellow fluorescent Protein “Venus”[4] (Part Bba_I757008) results in green fluorescence[2].
AASequence:
KNGIKANFKI RHNIEDGSVQ LADHYQQNTP IGDGPVLLPD NHYLSTQSKL SKDPNEKRDH
gene sequence:
MVLLEFVTAA GITLGMDELY KAAGAACGGC ATCAAGGCCA ACTTCAAGAT CCGGCACAAC ATCGAGGATG GCAGCGTGCA GCTGGCCGAT CACTACCAGC AGAACACCCC CATCGGCGAC GGCCCCGTGC TGCTGCCCGA CAACCACTAC CTGAGCACCC AGAGCAAGCT GTCCAAGGAC CCCAACGAGA AGCGGGACCA CATGGTGCTG CTGGAATTTG TGACAGCCGC CGGAATCACC CTGGGCATGG ACGAGCTGTA CAAG

• Split-Cerulean-N-CFP
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157006 BBa_K157006]
Description:
N-terminal fragment (Amino acids1-155) of the monomeric[5] cyan fluorescent protein variant “Cerulean”, designed for fusion/BiFC[1,3]. Reassembly with Split-Cerulean-cCFP (Part Bba_K157005) generates the complete, working CFP “Cerulean”, whereas combination with the C-terminal fragment (Part Bba_K157007) of the monomeric yellow fluorescent Protein “Venus”[4] (Part Bba_I757008) results in green fluorescence[2].
AASequence:
MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTLGVQCFA RYPDHMKRHD FFKSAMPEGY VQERTIFFKD DGNYKTRAEV KFEGDTLVNR IELKGIDFKE DGNILGHKLE YNAISDNVYI TADKQ
gene sequence:
ATGAGCAAGG GCGAGGAACT GTTCACCGGC GTGGTGCCCA TCCTGGTGGA GCTGGACGGC GACGTGAACG GCCACAAGTT CAGCGTGTCC GGCGAGGGCG AAGGCGACGC CACCTACGGC AAGCTGACCC TGAAGTTCAT CTGCACCACC GGCAAGCTGC CCGTGCCCTG GCCCACCCTG GTGACCACCC TGGGCGTGCA GTGCTTCGCC AGATACCCCG ACCACATGAA GCGGCACGAT TTCTTCAAGA GCGCCATGCC CGAGGGCTAC GTGCAGGAAC GGACCATCTT CTTCAAGGAC GACGGCAACT ACAAGACCAG AGCCGAAGTG AAGTTCGAGG GCGACACACT GGTGAACCGG ATCGAGCTGA AGGGCATCGA CTTCAAAGAG GACGGCAATA TCCTGGGCCA CAAGCTGGAA TACAACGCCA TCAGCGACAA CGTGTACATC ACCGCCGACA GCAG

• Split-Venus-C-YFP
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157007 BBa_K157007]
Description:
C-terminal fragment (Amino acids157-237) of the monomeric[5] yellow fluorescent protein variant “Venus” [4], designed for fusion/BiFC[1,3]. Reassembly with Part Bba_K157008 (Split-Venus-nYFP) generates the complete, working YFP “Venus”, whereas combination with the n-terminal fragment (Part Bba_K157006) of the monomeric cyan fluorescent Protein “CeruleanR206K” (Part Bba_I757009) results in green fluorescence[2].
AASequence:
KNGIKANFKI RHNIEDGGVQ LADHYQQNTP IGDGPVLLPD NHYLSYQSKL SKDPNEKRDH MVLLEFVTAA GITHGMDELY K
gene sequence:
AAGAACGGCA TCAAGGCCAA CTTCAAGATC CGGCACAACA TCGAGGACGG CGGCGTGCAG CTGGCCGACC ACTACCAGCA GAACACCCCC ATCGGCGACG GCCCCGTGCT GCTGCCCGAC AACCACTACC TGAGCTACCA GAGCAAGCTG TCCAAGGACC CCAACGAGAA GCGGGACCAC ATGGTGCTGC TGGAATTTGT GACAGCCGCC GGAATCACCC ACGGCATGGA CGAGCTGTAC AAG

• Split-Venus-N-YFP
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157008 BBa_K157008]
Description:
N-terminal fragment (Amino acids1-156) of the monomeric[5] yellow fluorescent protein variant “Venus” [4], designed for fusion/BiFC[1,3]. Reassembly with Part Bba_K157007 (Split-Venus-cYFP) generates the complete, working YFP “Venus”, whereas combination with the C-terminal fragment (Part Bba_K157005) of the monomeric cyan fluorescent Protein “CeruleanR206K” (Part Bba_I757009) results in green fluorescence[2].
AASequence:
MVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY GKLTLKLICT TGKLPVPWPT LVTTLYLQCF ARYPDHMKQH DFFKSAMPEG YVQERTIFFK DDGNYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNYNSHNVY ITADKQ
gene sequence:
ATGGTGTCCA AGGGCGAGGA ACTGTTCACC GGCGTGGTGC CCATCCTGGT GGAGCTGGAC GGCGACGTGA ACGGCCACAA GTTCAGCGTG TCCGGCGAGG GCGAAGGCGA CGCCACCTAC GGCAAGCTGA CCCTGAAGCT GATCTGCACC ACCGGCAAGC TGCCCGTGCC CTGGCCCACC CTGGTGACCA CCCTGTACCT CCAGTGCTTC GCCAGATACC CCGACCACAT GAAGCAGCAC GATTTCTTCA AGAGCGCCAT GCCCGAGGGC TACGTGCAGG AACGGACCAT CTTCTTCAAG GACGACGGCA ACTACAAGAC CAGAGCCGAA GTGAAGTTCG AGGGCGACAC ACTGGTGAAC CGGATCGAGC TGAAGGGCAT CGACTTCAAA GAGGACGGCA ATATCCTGGG CCACAAGCTG GAATACAACT ACAACAGCCA CAACGTGTAC ATCACCGCCG ACAAGCAG

Source (all split-fluorophores):
Gene synthesis by GeneArt, optimized for expression in homo sapiens.
References (all split-fluorophores):
- [1] Chang-Deng Hu, Yurii Chinenov, Tom K. Kerppola: ”Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation”, Molecular Cell, Vol. 9, 789–798, April, 2002
- [2] Chang Deng Hu, Tom K. Kerppola: “Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis”, Nat Biotechnol. 2003 May; 21(5):539-545 (doi:10. 1038/nbt816)
-[3] Tom K. Kerppola: “Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells”, Nat Protoc. 2006;1(3):1278-1286 (doi:10.1038/nprot.2006.201)
-[4]Nagai, T. et al. “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications” J. Biol. Chem. 276, 29188-29194, 2001
-[5]Roger Y. Tsien et al. „Creating new fluorescent probes for cell biology“, Nature Biotechnology Reviews, Vol. 3, 906-918, 2002

• Split-Fluo-Linker
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157009 BBa_K157009]
description:
Originally, this linker was used for fusion to the N-terminus of the C-terminal half of split fluorophores: Protein interactions can be examined by BiFC[1] using complementary, non-fluorescent fragments of fluorophores[2]. Therefore, it is essential that the C-terminal fragment of the fluorophore is not restricted too much in its mobility. The linker allows orientation and adaption of the C-terminal fragment to the N-terminal fragment of the split fluorophore and, thus, the reassembly of a working fluorescent protein. It has already been used in BiFC assays and is known to serve this purpose well[3]; anyway, another, more flexible linker that could be used instead is our “GGGGS-linker” (Part Bba_K157010).
References:
- [1] Chang-Deng Hu, Yurii Chinenov, Tom K. Kerppola: ”Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation”, Molecular Cell, Vol. 9, 789–798, April, 2002
- [2] Chang Deng Hu, Tom K. Kerppola: “Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis”, Nat Biotechnol. 2003 May; 21(5):539-545 (doi:10. 1038/nbt816)
-[3] Tom K. Kerppola: “Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells”, Nat Protoc. 2006;1(3):1278-1286 (doi:10.1038/nprot.2006.201)
source:
Gene synthesis by ATG:biosynthetics, optimized for expression in homo sapiens by iGEM-Team Freiburg 2008.
AA Sequence:
RPACKIPNDL KQKVMNH
gene sequence:
CGACCAGCCT GTAAGATTCC AAATGACCTG AAGCAGAAAG TTATGAATCA C

• GGGGS-LINKER_BCR-Transmem.
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157010 BBa_K157010]
Description:
Helical single-span transmembrane region of the B-Cell-Receptor with a flexible 15 aa Linker at the N-terminus. Designed for fusion to proteins or peptides that are to be presented at the cells surface; signal peptide for membrane integration (e.g. part Bba_K157001) required at the N-terminus of the whole construct!

• His-Tag (improved)
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157011 BBa_K157011]
See Part BBa_I757013 (Freiburg 2007)

• Strep-Tag II (improved)
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157012 BBa_K157012]
See Part BBa_I757014 (Freiburg 2007)

• GGGS-linker (pMA)
  

Part Name:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K157013 BBa_K157013]
Description:
A flexible, 15 aa long linker designed for fusion to/of proteins or peptides via in frame cloning. Consists of Glycine and Serine only.
source:
Gene synthesis by ATG:biosynthetics, optimized for expression in homo sapiens by iGEM-Team Freiburg 2008.
AA sequence:
GGGSGGGSGG GSGGG
gene sequence:
GGTGGAGGAG GTTCTGGAGG CGGTGGAAGT GGTGGCGGAG GTAGC

composite parts

Part BBa_K157014 - BBa_K157039, Part BBa_K157040 (modified expression plasmid with CMV-promoter(for expression in human cells) and BBa_K157041 (BBa_K157040 + CFP)