Difference between revisions of "Part:BBa K1080011:Design"

Revision as of 04:47, 24 September 2013

ChlI2

Design Notes

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

ChlI2 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: Three PstI sites at 368, 758, 920. No EcoRI, XbaI or SpeI sites. Tag needs to be removed to work.

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

TTGCCATCCACGAAAGCCGCGAAGAAGCCGAACTTCCCGTTCGTCAAGATTCAGGGCCAG

GAGGAGATGAAGCTTGCACTGCTGCTGAACGTGGTCGACCCCAACATCGGCGGAGTGCTT

ATTATGGGTGACCGCGGCACTGCCAAGTCGGTCGCGGTCCGCGCCCTGGTGGATATGCTT

CCCGACATTGACGTGGTTGAGGGCCACGCCTTCAACAGCTCCCCCACCGACCCCAAGTTC

ATGGGCCCCGACACCCTGCAGCGCTTCCGCAACGGCGAGAAGCTGCCCACCGTCCGCATG

CGGACCCCCCTGGTGGAGCTGCCTCTGGGCGCCACCGAGGACCGCATCTGCGGCACCATC

GACATCGAGAAGGCGCTGACGCAGGGCATCAAGGCCTACGAGCCCGGCCTGCTGGCCAAG

GCCAACCGCGGCATCCTGTATGTGGACGAGGTGAACCTGCTGGATGATGGCCTGGTTGAT

GTCGTGCTGGACTCGTCGGCTAGCGGCCTGAACACTGTGGAGCGTGAGGGTGTGTCCATT

GTGCACCCTGCCCGCTTCATCATGATTGGCTCAGGCAACCCCCAGGAGGGTGAGCTGCGC

CCGCAGCTGCTGGATCGCTTCGGCATGAGCGTCAACGTGGCCACGCTGCAGGACACCAAG

CAGCGCACGCAGCTGGTGCTGGACCGGCTTGCGTACGAGGCGGACCCTGACGCATTTGTG

GACTCGTGCAAGGCCGAGCAGACGGCGCTCACGGACAAGCTGGAGGCGGCCCGCCAGCGC

CTGCGGTCCGTCAAGATCAGCGAGGAGCTGCAGATCCTGATCTCGGACATTTGCTCGCGC

CTGGATGTGGATGGCCTGCGCGGTGACATTGTGATCAACCGCGCCGCCAAGGCGCTTGTG

GCCTTCGAGGGCCGCACCGAGGTGACCACGAATGACGTGGAGCGCGTCATCTCGGGCTGC

CTCAACCACCGCCTGCGCAAGGACCCGCTGGACCCCATTGACAACGGCACCAAGGTGGCC

ATCCTGTTCAAGCGCATGACCGACCCCGAGATCATGAAGCGCGAGGAGGAGGCCAAGAAG

AAGCGCGAGGAGGCGGCCGCCAAGGCCAAGGCGGAGGGCAAGGCGGACCGCCCCACGGGC

GCCAAGGCTGGCGCCTGGGCTGGCTTGCCCCCTCGTCGGTAAAAAAGGCTGGTGTAGGCC

TGTCGGGTCGTGTTAAAGGTTGCTGCGTGAACGTGTAAGTGTGACAGTGTGCCGGTATGT

GTGTGTATACATGTGTTGCGGTGTGCTTTTGTGGCGGTACATGGTGATGACTGAGCGGGT

GGGACAGAGCACGGTTAACTGACGAGG

Amino acid sequence

MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDHPFTLPST KAAKKPNFPF VKIQGQEEMK

LALLLNVVDP NIGGVLIMGD RGTAKSVAVR ALVDMLPDID VVEGDAFNSS PTDPKFMGPD

TLQRFRNGEK LPTVRMRTPL VELPLGATED RICGTIDIEK ALTQGIKAYE PGLLAKANRG

ILYVDEVNLL DDGLVDVVLD SSASGLNTVE REGVSIVHPA RFIMIGSGNP QEGELRPQLL

DRFGMSVNVA TLQDTKQRTQ LVLDRLAYEA DPDAFVDSCK AEQTALTDKL EAARQRLRSV

KISEELQILI SDICSRLDVD GLRGDIVINR AAKALVAFEG RTEVTTNDVE RVISGCLNHR

LRKDPLDPID NGTKVAILFK RMTDPEIMKR EEEAKKKREE AAAKAKAEGK ADRPTGAKAG

AWAGLPPRR

References and documentation are available.

Please note the modified algorithm for extinction coefficient.

Number of amino acids: 429

Molecular weight: 47061.7

Theoretical pI: 5.94

Amino acid composition:

Ala (A) 39 9.1% Arg (R) 32 7.5% Asn (N) 14 3.3% Asp (D) 38 8.9% Cys (C) 4 0.9% Gln (Q) 14 3.3% Glu (E) 28 6.5% Gly (G) 34 7.9% His (H) 9 2.1% Ile (I) 22 5.1% Leu (L) 43 10.0% Lys (K) 28 6.5% Met (M) 13 3.0% Phe (F) 11 2.6% Pro (P) 23 5.4% Ser (S) 18 4.2% Thr (T) 23 5.4% Trp (W) 1 0.2% Tyr (Y) 4 0.9% Val (V) 31 7.2%

Asx (B) 0 0.0% Glx (Z) 0 0.0% Xaa (X) 0 0.0%

Total number of negatively charged residues (Asp + Glu): 66 Total number of positively charged residues (Arg + Lys): 60

Atomic composition:

Carbon C 2046 Hydrogen H 3352 Nitrogen N 600 Oxygen O 635 Sulfur S 17

Formula: C2046H3352N600O635S17 Total number of atoms: 6650

Extinction coefficients:

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

Ext. coefficient 11710 Abs 0.1% (=1 g/l) 0.249, assuming ALL Cys residues appear as half cystines

Ext. coefficient 11460 Abs 0.1% (=1 g/l) 0.244, assuming NO Cys residues appear as half cystines

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 31.25 This classifies the protein as stable.

Aliphatic index: 89.14

Grand average of hydropathicity (GRAVY): -0.396

Source

Chlamydomonas reinhardtii

References