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

Revision as of 10:33, 25 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:

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

Amino acid sequence

MVAATAAPQE VEGFKVMRDG IKVASDETLL TPRFYTTDFD EMERLFSLEL NKNMDMEEFE
AMLNEFKLDY NQRHFVRNET FKEAAEKIQG PTRKIFIEFL ERSCTAEFSG FLLYKELGRR
LKATNPVVAE IFTLMSRDEA RHAGFLNKAM SDFNLALDLG FLTKNRKYTF FKPKFIFYAT
YLSEKIGYWR YISIYRHLQR NPDNQLYPLF EYFENWCQDE NRHGDFFTAV LKARPEMVND
WAAKLWSRFF CLSVYITMYL NDHQRDAFYS SLGLNTTQFN QHVIIETNKS TERIFPAVPD
VENPEFFRRM DLLVKYNAQL VNIGSMNLPS PIKAIMKAPI LERMVAEVFQ VFIMTPKESG
SYDLDANKTA LVY

References and documentation are available. Please note the modified algorithm for extinction coefficient.

Number of amino acids: 373
Molecular weight: 43873.3
Theoretical pI: 5.98
Amino acid composition: Ala (A) 28 7.5% Arg (R) 23 6.2% Asn (N) 23 6.2% Asp (D) 19 5.1% Cys (C) 3 0.8% Gln (Q) 11 2.9% Glu (E) 31 8.3% Gly (G) 12 3.2% His (H) 6 1.6% Ile (I) 18 4.8% Leu (L) 35 9.4% Lys (K) 23 6.2% Met (M) 15 4.0% Phe (F) 33 8.8% Pro (P) 15 4.0% Ser (S) 17 4.6% Thr (T) 21 5.6% Trp (W) 4 1.1% Tyr (Y) 17 4.6% Val (V) 19 5.1% 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): 50 Total number of positively charged residues (Arg + Lys): 46
Atomic composition:
Carbon C 1999 Hydrogen H 3041 Nitrogen N 515 Oxygen O 563 Sulfur S 18
Formula: C1999H3041N515O563S18 Total number of atoms: 6136
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 47455 Abs 0.1% (=1 g/l) 1.082, assuming all pairs of Cys residues form cystines

Ext. coefficient 47330 Abs 0.1% (=1 g/l) 1.079, 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 36.26 This classifies the protein as stable.

Aliphatic index: 77.69
Grand average of hydropathicity (GRAVY): -0.313

Source

Chlamydomonas reinhardtii

===References===

@@ Line 11: / Line 11: @@
 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
@@ Line 17: / Line 17: @@
 "ProtParam" at http://au.expasy.org/tools
-One PstI site at 740. No EcoRi, SpeI or XbaI
-<b>DNA-Sequence:</b>
-<b><FONT FACE="courier">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</FONT></b>
-<FONT FACE="courier">CAGGCCAGCGGGAATGAGCAGCAGTCTCGCCGGGTCCGTGTGGCTGCTACCGCTGCTCCCCAGGAGG
-TTGAGGGCTTCAAGGTTATGCGCGACGGCATCAAGGTTGCTTCCGATGAGACCCTGCTCACTCCTCG
-CTTCTACACCACCGACTTCGATGAGATGGAGCGCCTGTTCAGCCTGGAGCTGAACAAGAACATGGAC
-ATGGAGGAGTTCGAGGCGATGCTGAACGAGTTCAAGCTGGACTACAACCAGCGCCACTTCGTCCGCA
-ACGAGACCTTCAAGGAGGCCGCGGAGAAGATTCAGGGCCCCACCCGCAAGATTTTCATTGAGTTCCT
-GGAGCGCTCGTGCACTGCTGAGTTCTCGGGCTTCCTGCTCTACAAGGAGCTGGGTCGCCGCCTGAAG
-GCCACGAACCCTGTGGTCGCGGAGATCTTCACCCTGATGTCCCGTGACGAGGCCCGCCACGCCGGCT
-TCCTGAACAAGGCCATGTCCGACTTCAACCTGGCTCTGGACCTGGGTTTCCTGACCAAGAACCGCAA
-GTACACCTTCTTCAAGCCCAAGTTCATCTTCTACGCCACTTACCTGTCGGAGAAGATTGGCTACTGG
-CGCTACATTTCTATCTACCGCCACCTGCAGCGCAACCCCGACAACCAGCTGTACCCGCTGTTCGAGT
-ACTTCGAGAACTGGTGCCAGGACGAGAACCGCCACGGTGACTTCTTCACCGCCGTGCTGAAGGCGCG
-GCCGGAGATGGTCAACGACTGGGCTGCCAAGCTGTGGTCGCGCTTTTTCTGCCTGTCGGTGTACATC
-ACCATGTACCTGAACGACCACCAGCGCGACGCCTTCTACAGCTCTCTGGGCCTGAACACCACCCAGT
-TCAACCAGCACGTGATCATCGAGACCAACAAGTCTACTGAGCGTATCTTCCCCGCTGTGCCGGATGT
-GGAGAACCCCGAGTTCTTCCGCCGCATGGACCTGCTGGTCAAGTACAACGCCCAGCTGGTGAACATC
-GGCAGCATGAACCTGCCCAGCCCCATCAAGGCGATCATGAAGGCGCCCATTCTGGAGCGCATGGTCG
-CTGAGGTGTTCCAGGTGTTCATCATGACCCCTAAGGAGTCGGGCTCGTACGACCTGGATGCCAACAA
-GACCGCTCTGGTCTACTAAGCGGCTGGCGTACTAGCCTGCGGGAGCTGGGCGGCTGGATGTCGGAGT
-TTTGAGAGTGCTTTGGAGCTCGGCGGCGAGCAGCAGCTGTGTGTGGCAGCGTGTGTGTGCAGCAGCA
-AGTGTAGGTATACAGCCAGTGGCGCTGCTCGCTGGCGGCGCGGTGTCGTGTTGGGCGCCGTCCGACA
-GCGCCGCGGCGGCCCGCTGCCGTAGTCGACGCGGGTTGGTCACTCATCCAGGAGACGACCTCTCGAG
-TAGCTGATAGCTTGAGATGAGGGTTCTCTATGCGC</FONT>
+<b>Amino acid sequence</b>
+<b><FONT FACE="courier">MVAATAAPQE VEGFKVMRDG IKVASDETLL TPRFYTTDFD EMERLFSLEL NKNMDMEEFE<br> AMLNEFKLDY NQRHFVRNET FKEAAEKIQG PTRKIFIEFL ERSCTAEFSG FLLYKELGRR<br> LKATNPVVAE IFTLMSRDEA RHAGFLNKAM SDFNLALDLG FLTKNRKYTF FKPKFIFYAT<br> YLSEKIGYWR YISIYRHLQR NPDNQLYPLF EYFENWCQDE NRHGDFFTAV LKARPEMVND<br> WAAKLWSRFF CLSVYITMYL NDHQRDAFYS SLGLNTTQFN QHVIIETNKS TERIFPAVPD<br> VENPEFFRRM DLLVKYNAQL VNIGSMNLPS PIKAIMKAPI LERMVAEVFQ VFIMTPKESG<br> SYDLDANKTA LVY  </FONT>
-<b>PROTEIN Sequence:</b>
+References and documentation are available.
+Please note the modified algorithm for extinction coefficient.
-<FONT FACE="courier">MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDHPFTQASGNEQQSRRVRVAATAAPQEVE
-GFKVMRDGIKVASDETLLTPRFYTTDFDEMERLFSLELNKNMDMEEFEAMLNEFKLDYNQ
-RHFVRNETFKEAAEKIQGPTRKIFIEFLERSCTAEFSGFLLYKELGRRLKATNPVVAEIF
-TLMSRDEARHAGFLNKAMSDFNLALDLGFLTKNRKYTFFKPKFIFYATYLSEKIGYWRYI
-SIYRHLQRNPDNQLYPLFEYFENWCQDENRHGDFFTAVLKARPEMVNDWAAKLWSRFFCL
-SVYITMYLNDHQRDAFYSSLGLNTTQFNQHVIIETNKSTERIFPAVPDVENPEFFRRMDL
-LVKYNAQLVNIGSMNLPSPIKAIMKAPILERMVAEVFQVFIMTPKESGSYDLDANKTALV
-Y</FONT>
+--------------------------------------------------------------------------------
+Number of amino acids: 373
-Number of amino acids: 421
+Molecular weight: 43873.3
-Molecular weight: 49365.1
+Theoretical pI: 5.98
-Theoretical pI: 6.25
+Amino acid composition: Ala (A)  28	  7.5%
+Arg (R)  23	  6.2%
+Asn (N)  23	  6.2%
+Asp (D)  19	  5.1%
+Cys (C)   3	  0.8%
+Gln (Q)  11	  2.9%
+Glu (E)  31	  8.3%
+Gly (G)  12	  3.2%
+His (H)   6	  1.6%
+Ile (I)  18	  4.8%
+Leu (L)  35	  9.4%
+Lys (K)  23	  6.2%
+Met (M)  15	  4.0%
+Phe (F)  33	  8.8%
+Pro (P)  15	  4.0%
+Ser (S)  17	  4.6%
+Thr (T)  21	  5.6%
+Trp (W)   4	  1.1%
+Tyr (Y)  17	  4.6%
+Val (V)  19	  5.1%
+Pyl (O)   0	  0.0%
+Sec (U)   0	  0.0%
-Amino acid composition:
+ (B)   0	  0.0%
-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%
+ (Z)   0	  0.0%
+ (X)   0	  0.0%
-Total number of negatively charged residues (Asp + Glu): 57
+Total number of negatively charged residues (Asp + Glu): 50
-Total number of positively charged residues (Arg + Lys): 52
+Total number of positively charged residues (Arg + Lys): 46
 Atomic composition:
-Carbon      C	      2222
+Carbon      C	      1999
-Hydrogen    H	      3379
+Hydrogen    H	      3041
-Nitrogen    N	       599
+Nitrogen    N	       515
-Oxygen      O	       638
+Oxygen      O	       563
-Sulfur      S	        21
+Sulfur      S	        18
-Formula: C2222H3379N599O638S21
+Formula: C1999H3041N515O563S18
-Total number of atoms: 6859
+Total number of atoms: 6136
 Extinction coefficients:
@@ Line 111: / Line 78: @@
 Extinction coefficients are in units of  M-1 cm-1, at 280 nm measured in water.
-Ext. coefficient    48945
+Ext. coefficient    47455
-Abs 0.1% (=1 g/l)   0.991, assuming all pairs of Cys residues form cystines
+Abs 0.1% (=1 g/l)   1.082, assuming all pairs of Cys residues form cystines
-Ext. coefficient    48820
+Ext. coefficient    47330
-Abs 0.1% (=1 g/l)   0.989, assuming all Cys residues are reduced
+Abs 0.1% (=1 g/l)   1.079, assuming all Cys residues are reduced
 Estimated half-life:
@@ Line 130: / Line 97: @@
 Instability index:
-The instability index (II) is computed to be 37.56
+The instability index (II) is computed to be 36.26
 This classifies the protein as stable.
-Aliphatic index: 70.93
+Aliphatic index: 77.69
-Grand average of hydropathicity (GRAVY): -0.477
+Grand average of hydropathicity (GRAVY): -0.313
 ===Source===