Difference between revisions of "Part:BBa K1080002:Design"

Revision as of 05:23, 23 September 2013

ChlD

Design Notes

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

ChlD 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

Note: No XbaI, EcoRI, PstI or SpeI sites

ATG CGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT GGT GGA

CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG GAT CAT CCC TTC ACC

CTGCGCGCCATGAAGGTGTCTGAGGAGGACTCCAAGGGCTTCGATGCGGATGTGTCGACCCGCCTGGCCCGCTCG TACCCTCTGGCGGCCGTGGTGGGCCAGGACAACATCAAGCAGGCGCTGCTGCTGGGCGCCGTGGACACCGGGCTG GGCGGCATCGCCATCGCCGGTCGCCGCGGTACCGCCAAGTCCATCATGGCTCGCGGCCTGCACGCTCTGCTGCCG CCCATTGAGGTGGTGGAGGGCAGCATCTGCAACGCCGACCCCGAGGACCCCCGCTCCTGGGAGGCTGGCCTGGCT GAGAAGTATGCGGGCGGCCCTGTGAAGACCAAGATGCGCTCGGCGCCGTTTGTGCAGATCCCTCTGGGTGTGACT GAGGACCGCTTGGTGGGCACTGTGGACATTGAGGCGTCCATGAAGGAGGGCAAGACTGTGTTCCAGCCCGGCCTG CTGGCTGAGGCGCACCGCGGCATCCTGTACGTGGACGAGATCAACCTGCTGGATGACGGCATTGCCAACCTGCTG CTGTCCATCCTGTCGGACGGAGTCAACGTGGTGGAGCGCGAGGGCATCTCCATCAGCCACCCCTGCCGGCCGCTG CTGATTGCCACCTACAACCCCGAGGAGGGCCCTCTGCGTGAGCACCTGCTGGACCGCATCGCCATTGGCCTCAGC GCCGACGTCCCCAGCACCAGCGACGAGCGCGTCAAGGCCATTGACGCAGCCATCCGCTTCCAGGACAAGCCGCAG GACACTATTGACGACACCGCGGAGCTCACCGACGCCCTGCGCACCTCGGTCATCCTGGCTCGCGAGTACCTGAAG GACGTGACCATCGCGCCGGAGCAGGTGACCTACATTGTGGAGGAGGCGCGCCGCGGCGGAGTCCAGGGGCACCGC GCGGAGCTGTACGCGGTCAAGTGTGCCAAGGCGTGTGCGGCTCTGGAGGGCCGTGAGCGTGTGAACAAGGATGAC CTGCGCCAGGCCGTGCAGCTGGTCATCCTGCCGCGCGCCACCATCCTGGACCAGCCCCCGCCCGAGCAGGAGCAG CCCCCGCCGCCGCCCCCGCCCCCTCCCCCGCCGCCGCCGCAGGACCAAATGGAGGACGAGGACCAGGAGGAGAAG GAGGACGAGAAGGAGGAGGAGGAGAAGGAGAACGAGGACCAGGACGAGCCCGAGATCCCTCAGGAGTTCATGTTT GAGTCCGAGGGCGTCATCATGGACCCCTCCATCCTCATGTTCGCGCAGCAGCAGCAGCGCGCGCAGGGCCGCTCC GGCCGCGCCAAGACGCTCATCTTCAGCGACGACCGCGGCCGCTACATCAAGCCCATGCTGCCCAAGGGTGACAAG GTCAAGCGCCTGGCAGTGGACGCCACGCTTCGCGCCGCCGCGCCCTACCAGAAGATTCGCCGGCAGCAGGCCATC AGCGAGGGCAAGGTGCAGCGCAAGGTGTACGTGGACAAGCCAGACATGCGCTCCAAGAAGCTGGCCCGCAAGGCC GGTGCGCTGGTGATTTTTGTTGTGGACGCGTCCGGCTCCATGGCTCTGAACCGCATGAGCGCCGCCAAGGGCGCC TGCATGCGCCTGCTGGCTGAGTCGTACACCAGCCGCGACCAGGTGTGCCTCATCCCCTTCTACGGCGACAAGGCC GAGGTGCTGCTGCCGCCCTCCAAGTCCATCGCCATGGCCCGCCGCCGCCTGGACTCGCTGCCCTGCGGCGGCGGC TCGCCCCTTGCGCACGGCCTGTCCACGGCGGTACGTGTGGGCATGCAGGCCAGCCAGGCGGGCGAGGTGGGCCGC GTCATGATGGTGCTCATCACGGACGGCCGCGCCAACGTCAGCCTGGCCAAGTCCAACGAGGACCCCGAGGCGCTC AAGCCCGACGCGCCCAAGCCCACCGCCGACTCGCTGAAGGACGAGGTGCGCGACATGGCCAAGAAGGCCGCGTCC GCCGGCATCAACGTGCTTGTCATTGACACGGAGAACAAGTTCGTGAGCACCGGCTTTGCGGAGGAGATCTCCAAG GCAGCGCAGGGCAAGTACTACTACCTGCCCAACGCCAGCGACGCCGCCATCGCGGCGGCCGCGTCCGGCGCCATG GCCGCGGCCAAGGGCGGCTACTAGGTGCCGAGTGACTGAGGTGGCAAGGTGCAGTGGCGGCGGAGGCAGTTGTGC TGGGGTGGCAAGGCGGACAGGCGAAGCTGGTGGGTTGCGACGAGGAGGAGGTGCACGTGCACGCGTAACATAAGA AGAACAGTGGGAGGACAGGTAGCGTGACTTGACTGGGACGAGGAGCGTACTGATGTGTGGCGTGTGTTGGTATGT
GAGCGTTACCCCTCC

Protein sequence:

       10         20         30         40         50         60 

MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDHPFTLRAM KVSEEDSKGF DADVSTRLAR

       70         80         90        100        110        120 

SYPLAAVVGQ DNIKQALLLG AVDTGLGGIA IAGRRGTAKS IMARGLHALL PPIEVVEGSI

      130        140        150        160        170        180 

CNADPEDPRS WEAGLAEKYA GGPVKTKMRS APFVQIPLGV TEDRLVGTVD IEASMKEGKT

      190        200        210        220        230        240 

VFQPGLLAEA HRGILYVDEI NLLDDGIANL LLSILSDGVN VVEREGISIS HPCRPLLIAT

      250        260        270        280        290        300 

YNPEEGPLRE HLLDRIAIGL SADVPSTSDE RVKAIDAAIR FQDKPQDTID DTAELTDALR

      310        320        330        340        350        360 

TSVILAREYL KDVTIAPEQV TYIVEEARRG GVQGHRAELY AVKCAKACAA LEGRERVNKD

      370        380        390        400        410        420 

DLRQAVQLVI LPRATILDQP PPEQEQPPPP PPPPPPPPPQ DQMEDEDQEE KEDEKEEEEK

      430        440        450        460        470        480 

ENEDQDEPEI PQEFMFESEG VIMDPSILMF AQQQQRAQGR SGRAKTLIFS DDRGRYIKPM

      490        500        510        520        530        540 

LPKGDKVKRL AVDATLRAAA PYQKIRRQQA ISEGKVQRKV YVDKPDMRSK KLARKAGALV

      550        560        570        580        590        600 

IFVVDASGSM ALNRMSAAKG ACMRLLAESY TSRDQVCLIP FYGDKAEVLL PPSKSIAMAR

      610        620        630        640        650        660 

RRLDSLPCGG GSPLAHGLST AVRVGMQASQ AGEVGRVMMV LITDGRANVS LAKSNEDPEA

      670        680        690        700        710        720 

LKPDAPKPTA DSLKDEVRDM AKKAASAGIN VLVIDTENKF VSTGFAEEIS KAAQGKYYYL

      730        740 

PNASDAAIAA AASGAMAAAK GGY

References and documentation are available.

Please note the modified algorithm for extinction coefficient.

Number of amino acids: 743

Molecular weight: 80527.5

Theoretical pI: 5.37

Amino acid composition: Ala (A) 89 12.0% Arg (R) 49 6.6% Asn (N) 14 1.9% Asp (D) 57 7.7% Cys (C) 7 0.9% Gln (Q) 33 4.4% Glu (E) 55 7.4% Gly (G) 56 7.5% His (H) 13 1.7% Ile (I) 40 5.4% Leu (L) 62 8.3% Lys (K) 44 5.9% Met (M) 23 3.1% Phe (F) 13 1.7% Pro (P) 51 6.9% Ser (S) 44 5.9% Thr (T) 26 3.5% Trp (W) 1 0.1% Tyr (Y) 17 2.3% Val (V) 49 6.6% 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): 112 Total number of positively charged residues (Arg + Lys): 93

Atomic composition:

Carbon C 3504 Hydrogen H 5684 Nitrogen N 1008 Oxygen O 1102 Sulfur S 30

Formula: C3504H5684N1008O1102S30 Total number of atoms: 11328

Extinction coefficients:

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

Ext. coefficient 31205 Abs 0.1% (=1 g/l) 0.388, assuming all pairs of Cys residues form cystines

Ext. coefficient 30830 Abs 0.1% (=1 g/l) 0.383, 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 44.71 This classifies the protein as unstable.

Aliphatic index: 84.64

Grand average of hydropathicity (GRAVY): -0.392

Source

Chlamydomonas reinhardtii

References