Difference between revisions of "Part:BBa K1080005:Design"

(→‎Design Notes)
(→‎Design Notes)
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<b>DNA-Sequence:</b>
 
<b>DNA-Sequence:</b>
 
<b>ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGT
 
<b>ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGT
−
 
+
   GGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCATCCC
−
   GGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCATCCCT
+
   TTCACC<b>
−
 
+
−
   TCACC<b>
+
  
  

Revision as of 03:53, 24 September 2013


CTH1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 305
    Illegal BglII site found at 413
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 103
    Illegal BsaI.rc site found at 259
    Illegal BsaI.rc site found at 880


Design Notes

Incorporated sequence overlap for Gibson assembly and no GC rich region or restriction site in sequence


CTH1 Clone: DNA sequence from translation start site: Regions in BOLD are the sequence of the leader region in the pET100 plasmid. Translated the DNA sequence into a protein sequence using "Translate" at http://au.expasy.org/tools Then used the translated protein sequence to analyse the protein using "ProtParam" at http://au.expasy.org/tools

One PstI site at 740. No EcoRi, SpeI or XbaI

DNA-Sequence: ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGT 

  GGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCATCCC
  TTCACC<b>


CAGGCCAGCGGGAATGAGCAGCAGTCTCGCCGGGTCCGTGTGGCTGCTACCGCTGCTCCCCAGGAGGTTGAGGGCTTCAAGGTTATGCGCGACGGCATCAAGGTTGCTTCCGATGAGACCCTGCTCACTCCTCGCTTCTACACCACCGACTTCGATGAGATGGAGCGCCTGTTCAGCCTGGAGCTGAACAAGAACATGGACATGGAGGAGTTCGAGGCGATGCTGAACGAGTTCAAGCTGGACTACAACCAGCGCCACTTCGTCCGCAACGAGACCTTCAAGGAGGCCGCGGAGAAGATTCAGGGCCCCACCCGCAAGATTTTCATTGAGTTCCTGGAGCGCTCGTGCACTGCTGAGTTCTCGGGCTTCCTGCTCTACAAGGAGCTGGGTCGCCGCCTGAAGGCCACGAACCCTGTGGTCGCGGAGATCTTCACCCTGATGTCCCGTGACGAGGCCCGCCACGCCGGCTTCCTGAACAAGGCCATGTCCGACTTCAACCTGGCTCTGGACCTGGGTTTCCTGACCAAGAACCGCAAGTACACCTTCTTCAAGCCCAAGTTCATCTTCTACGCCACTTACCTGTCGGAGAAGATTGGCTACTGGCGCTACATTTCTATCTACCGCCACCTGCAGCGCAACCCCGACAACCAGCTGTACCCGCTGTTCGAGTACTTCGAGAACTGGTGCCAGGACGAGAACCGCCACGGTGACTTCTTCACCGCCGTGCTGAAGGCGCGGCCGGAGATGGTCAACGACTGGGCTGCCAAGCTGTGGTCGCGCTTTTTCTGCCTGTCGGTGTACATCACCATGTACCTGAACGACCACCAGCGCGACGCCTTCTACAGCTCTCTGGGCCTGAACACCACCCAGTTCAACCAGCACGTGATCATCGAGACCAACAAGTCTACTGAGCGTATCTTCCCCGCTGTGCCGGATGTGGAGAACCCCGAGTTCTTCCGCCGCATGGACCTGCTGGTCAAGTACAACGCCCAGCTGGTGAACATCGGCAGCATGAACCTGCCCAGCCCCATCAAGGCGATCATGAAGGCGCCCATTCTGGAGCGCATGGTCGCTGAGGTGTTCCAGGTGTTCATCATGACCCCTAAGGAGTCGGGCTCGTACGACCTGGATGCCAACAAGACCGCTCTGGTCTACTAAGCGGCTGGCGTACTAGCCTGCGGGAGCTGGGCGGCTGGATGTCGGAGTTTTGAGAGTGCTTTGGAGCTCGGCGGCGAGCAGCAGCTGTGTGTGGCAGCGTGTGTGTGCAGCAGCAAGTGTAGGTATACAGCCAGTGGCGCTGCTCGCTGGCGGCGCGGTGTCGTGTTGGGCGCCGTCCGACAGCGCCGCGGCGGCCCGCTGCCGTAGTCGACGCGGGTTGGTCACTCATCCAGGAGACGACCTCTCGAGTAGCTGATAGCTTGAGATGAGGGTTCTCTATGCGC


<b>PROTEIN Sequence:

MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFTQASGNEQQSRRVRVAATAAPQEVE GFKVMRDGIKVASDETLLTPRFYTTDFDEMERLFSLELNKNMDMEEFEAMLNEFKLDYNQ RHFVRNETFKEAAEKIQGPTRKIFIEFLERSCTAEFSGFLLYKELGRRLKATNPVVAEIF TLMSRDEARHAGFLNKAMSDFNLALDLGFLTKNRKYTFFKPKFIFYATYLSEKIGYWRYI SIYRHLQRNPDNQLYPLFEYFENWCQDENRHGDFFTAVLKARPEMVNDWAAKLWSRFFCL SVYITMYLNDHQRDAFYSSLGLNTTQFNQHVIIETNKSTERIFPAVPDVENPEFFRRMDL LVKYNAQLVNIGSMNLPSPIKAIMKAPILERMVAEVFQVFIMTPKESGSYDLDANKTALV Y


Number of amino acids: 421

Molecular weight: 49365.1

Theoretical pI: 6.25

Amino acid composition: Ala (A) 30 7.1% Arg (R) 28 6.7% Asn (N) 24 5.7% Asp (D) 25 5.9% Cys (C) 3 0.7% Gln (Q) 16 3.8% Glu (E) 32 7.6% Gly (G) 18 4.3% His (H) 13 3.1% Ile (I) 18 4.3% Leu (L) 36 8.6% Lys (K) 24 5.7% Met (M) 18 4.3% Phe (F) 34 8.1% Pro (P) 16 3.8% Ser (S) 21 5.0% Thr (T) 23 5.5% Trp (W) 4 1.0% Tyr (Y) 18 4.3% Val (V) 20 4.8% Pyl (O) 0 0.0% Sec (U) 0 0.0% (B) 0 0.0% (Z) 0 0.0% (X) 0 0.0%


Total number of negatively charged residues (Asp + Glu): 57 Total number of positively charged residues (Arg + Lys): 52

Atomic composition:

Carbon C 2222 Hydrogen H 3379 Nitrogen N 599 Oxygen O 638 Sulfur S 21

Formula: C2222H3379N599O638S21 Total number of atoms: 6859

Extinction coefficients:

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 48945 Abs 0.1% (=1 g/l) 0.991, assuming all pairs of Cys residues form cystines


Ext. coefficient 48820 Abs 0.1% (=1 g/l) 0.989, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is: 

                            30 hours (mammalian reticulocytes, in vitro).
                           >20 hours (yeast, in vivo).
                           >10 hours (Escherichia coli, in vivo).


Instability index:

The instability index (II) is computed to be 37.56 This classifies the protein as stable.


Aliphatic index: 70.93

Grand average of hydropathicity (GRAVY): -0.477

Source

Chlamydomonas reinhardtii

References