Coding

Part:BBa_K1159106

Designed by: Dong-Jiunn Jeffery TRUONG   Group: iGEM13_TU-Munich   (2013-09-16)

PhyB(2-908 AA)_36AALinker_C-TEV-Protease (First part for a light-switchable TEV-Protease) in RFC[25]

This parts encodes are fusion protein consisting the photosensitive core of PhyB (2-908 AA) at the N-terminus and the C-terminal half of TEV Protease at its C-terminus. Both parts are seperated by a 36 amino acids long flexible linker. This part is flanked by RFC[25] pre- and suffix to allow further protein fusions.

By expressing this part together with its interaction partner BBa_K1159107 or BBa_K1159108 that contains the part which interacts with PhyB (PIF3 or PIF6) and the N-terminal half of the splitted TEV Protease, they form together a light-switchable TEV Protease system which can be activated by red light and inactivated by far-red light.

Note: PhyB requires Phycocyanobilin or Phytochromobilin as a cofactor to fulfill its function as photoswitchable protein; you can reach it by adding exogenous Phycocyanobilin/Phytochromobilin in the medium or by inserting genes for the Phycocyanobilin/Phytochromobilin synthesis. See here for more information: [http://2012.igem.org/Team:TU_Munich/Project/Light_Switchable_Promoter#Biosynthesis_of_Phycocyanobilin Click here]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 487
    Illegal BamHI site found at 569
    Illegal XhoI site found at 520
    Illegal XhoI site found at 539
    Illegal XhoI site found at 2674
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2059
    Illegal BsaI site found at 2465
    Illegal SapI site found at 736
    Illegal SapI.rc site found at 3151

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
 ATGGCCGGCGTTTCCGGA ... CTCATGAATACCGGT
 ORF from nucleotide position -8 to 3201 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
1 
101 
201 
301 
401 
501 
601 
701 
801 
901 
1001 		MAGVSGVGGSGGGRGGGRGGEEEPSSSHTPNNRRGGEQAQSSGTKSLRPRSNTESMSKAIQQYTVDARLHAVFEQSGESGKSFDYSQSLKTTTYGSSVPE
QQITAYLSRIQRGGYIQPFGCMIAVDESSFRIIGYSENAREMLGIMPQSVPTLEKPEILAMGTDVRSLFTSSSSILLERAFVAREITLLNPVWIHSKNTG
KPFYAILHRIDVGVVIDLEPARTEDPALSIAGAVQSQKLAVRAISQLQALPGGDIKLLCDTVVESVRDLTGYDRVMVYKFHEDEHGEVVAESKRDDLEPY
IGLHYPATDIPQASRFLFKQNRVRMIVDCNATPVLVVQDDRLTQSMCLVGSTLRAPHGCHSQYMANMGSIASLAMAVIINGNEDDGSNVASGRSSMRLWG
LVVCHHTSSRCIPFPLRYACEFLMQAFGLQLNMELQLALQMSEKRVLRTQTLLCDMLLRDSPAGIVTQSPSIMDLVKCDGAAFLYHGKYYPLGVAPSEVQ
IKDVVEWLLANHADSTGLSTDSLGDAGYPGAAALGDAVCGMAVAYITKRDFLFWFRSHTAKEIKWGGAKHHPEDKDDGQRMHPRSSFQAFLEVVKSRSQP
WETAEMDAIHSLQLILRDSFKESEAAMNSKVVDGVVQPCRDMAGEQGIDELGAVAREMVRLIETATVPIFAVDAGGCINGWNAKIAELTGLSVEEAMGKS
LVSDLIYKENEATVNKLLSRALRGDEEKNVEVKLKTFSPELQGKAVFVVVNACSSKDYLNNIVGVCFVGQDVTSQKIVMDKFINIQGDYKAIVHSPNPLI
PPIFAADENTCCLEWNMAMEKLTGWSRSEVIGKMIVGEVFGSCCMLKGPDALTKFMIVLHNAIGGQDTDKFPFPFFDRNGKFVQALLTANKRVSLEGKVI
GAFCFLQIPSTGGGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSGTGKSMSSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHS
ASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMNTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 911 to 912, 949 to 950
Amino acid composition:
Ala (A)81 (7.6%)
Arg (R)48 (4.5%)
Asn (N)38 (3.6%)
Asp (D)55 (5.1%)
Cys (C)22 (2.1%)
Gln (Q)47 (4.4%)
Glu (E)60 (5.6%)
Gly (G)106 (9.9%)
His (H)23 (2.1%)
Ile (I)55 (5.1%)
Leu (L)84 (7.9%)
Lys (K)51 (4.8%)
Met (M)36 (3.4%)
Phe (F)43 (4.0%)
Pro (P)45 (4.2%)
Ser (S)102 (9.5%)
Thr (T)56 (5.2%)
Trp (W)14 (1.3%)
Tyr (Y)22 (2.1%)
Val (V)82 (7.7%)
Amino acid counting
Total number:1070
Positively charged (Arg+Lys):99 (9.3%)
Negatively charged (Asp+Glu):115 (10.7%)
Aromatic (Phe+His+Try+Tyr):102 (9.5%)
Biochemical parameters
Atomic composition:C5078H8016N1410O1566S58
Molecular mass [Da]:115735.5
Theoretical pI:5.86
Extinction coefficient at 280 nm [M-1 cm-1]:109780 / 111155 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges 
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.73)good (0.74)good (0.67)good (0.75)excellent (0.80)good (0.66)
Alignments (obtained from PredictProtein.org)
SwissProt:P14713 (99% identity on 915 AAs), P42497 (83% identity on 898 AAs)
TrEML:B0FWJ8 (99% identity on 898 AAs), Q5G893 (99% identity on 895 AAs), Q5G894 (99% identity on 898 AAs), Q5G899 (99% identity on 898 AAs), Q5G8A1 (99% identity on 898 AAs), Q5G8A3 (99% identity on 913 AAs), Q5G8A4 (99% identity on 895 AAs), Q5G8A5 (99% identity on 913 AAs), B0FWI4 (98% identity on 898 AAs), B0FWI5 (98% identity on 898 AAs)
PDB: -
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets)
Archaea:secreted (100%)
Bacteria:secreted (52%)
Eukarya:cytosol (39%)
Gene Ontology (reliability in brackets)
Molecular Function Ontology:GO:0009883 (70%), GO:0042802 (26%)
Biological Process Ontology:GO:0006350 (30%), GO:0006355 (29%)
 
Predicted features:
Disulfid bridges: -
Transmembrane helices: -
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Categories
Parameters
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