Coding

Part:BBa_K1159014

Designed by: Louise FUNKE   Group: iGEM13_TU-Munich   (2013-05-17)

Membrane-anchored Erythromycin Esterase Type II in RFC[25] N-Part

This part codes for a membrane-anchored version of Erythromycin Esterase Type II (EreB) for the chassis Physcomitrella patens. Please see subparts for for further description. This construct is flanked by RFC[25] N-part prefix and suffix. Note: This means only protein fusions to the C-terminus of this part is possible, adding promoters (typically RFC[10]) into the prefix or terminators/IRES (typically RFC[10]) into the suffix of this part is nevertheless possible.


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 295
    Illegal XhoI site found at 705
    Illegal XhoI site found at 1295
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1374
    Illegal BsaI.rc site found at 2292
    Illegal SapI.rc site found at 422

Protein data table for BioBrick BBa_K1159014 automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 25 N-Part using the stop codon in the suffix, so ACCGGT was added (in italics) to the 3' end: (underlined part encodes the protein)
 AACAATGCCTGGT ... CTCTACAAAACCGGT
 ORF from nucleotide position 5 to 2368 (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 		MPGEVAWWRPLFLIALMPIGVLSNAEGDALNTTGRFEEWVKDKHIPFKLNHPDDNYDDFKPLRKIIGDTRVVALGENSHFIKEFFLLRHTLLRFFIEDLG
FTTFAFEFGFAEGQIINNWIHGQGTDDEIGRFLKHFYYPEELKTTFLWLREYNKAAKEKITFLGIDIPRNGGSYLPNMEIVHDFFRTADKEALHIIDDAF
NIAKKIDYFSTSQAALNLHELTDSEKCRLTSQLARVKVRLEAMAPIHIEKYGIDKYETILHYANGMIYLDYNIQAMSGFISGGGMQGDMGAKDKYMADSV
LWHLKNPQSEQKVIVVAHNAHIQKTPILYDGFLSCLPMGQRLKNAIGDDYMSLGITSYSGHTAALYPEVDTKYGFRVDNFQLQEPNEGSVEKAISGCGVT
NSFVFFRNIPEDLQSIPNMIRFDSIYMKAELEKAFDGIFQIEKSSVSEVVYETGSAWSHPQFEKGENLYFQSGTGPGQPPFPPPPPFTPPPPQTPNGASG
ENSTGAIAGGVAAGAALLFAAPAIGFAWWRRRRPIEATGGGSGGGSGTGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK
LPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYI
MADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLSPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 33 to 34, 453 to 454, 474 to 475, 538 to 539, 548 to 549
Strep-tag II: 457 to 464
TEV cleavage site: 466 to 472
Amino acid composition:
Ala (A)51 (6.5%)
Arg (R)27 (3.4%)
Asn (N)38 (4.8%)
Asp (D)48 (6.1%)
Cys (C)5 (0.6%)
Gln (Q)26 (3.3%)
Glu (E)52 (6.6%)
Gly (G)76 (9.6%)
His (H)26 (3.3%)
Ile (I)53 (6.7%)
Leu (L)61 (7.7%)
Lys (K)50 (6.3%)
Met (M)18 (2.3%)
Phe (F)50 (6.3%)
Pro (P)43 (5.5%)
Ser (S)41 (5.2%)
Thr (T)44 (5.6%)
Trp (W)10 (1.3%)
Tyr (Y)31 (3.9%)
Val (V)38 (4.8%)
Amino acid counting
Total number:788
Positively charged (Arg+Lys):77 (9.8%)
Negatively charged (Asp+Glu):100 (12.7%)
Aromatic (Phe+His+Try+Tyr):117 (14.8%)
Biochemical parameters
Atomic composition:C3989H6057N1045O1169S23
Molecular mass [Da]:88094.7
Theoretical pI:5.60
Extinction coefficient at 280 nm [M-1 cm-1]:101190 / 101503 (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 (0.74)good (0.76)good (0.75)excellent (0.82)good (0.78)good (0.68)
Alignments (obtained from PredictProtein.org)
SwissProt:P05789 (100% identity on 418 AAs), P42212 (98% identity on 238 AAs)
TrEML:Q2V0Y9 (100% identity on 418 AAs), Q71RY9 (98% identity on 238 AAs)
PDB:2y0g (98% identity on 226 AAs), 1c4f (97% identity on 225 AAs)
Predictions (obtained from PredictProtein.org)
Subcellular Localization (reliability in brackets)
Archaea:cytosol (100%)
Bacteria:cytosol (62%)
Eukarya:cytosol (62%)
Gene Ontology (reliability in brackets)
Molecular Function Ontology: -
Biological Process Ontology:GO:0008218 (25%), GO:0046677 (25%)
 
Predicted features:
Disulfid bridges: -
Transmembrane helices:509 to 526 going inwards
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Categories
Parameters
None