Part:BBa_K1080011:Design
ChlI2
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 523
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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