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
TTGCCATCCACGAAAGCCGCGAAG
121 AAGCCGAACTTCCCGTTCGTCAAGATTCAGGGCCAGGAGGAGATGAAGCTTGCACTGCTG
181 CTGAACGTGGTCGACCCCAACATCGGCGGAGTGCTTATTATGGGTGACCGCGGCACTGCC
241 AAGTCGGTCGCGGTCCGCGCCCTGGTGGATATGCTTCCCGACATTGACGTGGTTGAGGGC
301 GACGCCTTCAACAGCTCCCCCACCGACCCCAAGTTCATGGGCCCCGACACCCTGCAGCGC
361 TTCCGCAACGGCGAGAAGCTGCCCACCGTCCGCATGCGGACCCCCCTGGTGGAGCTGCCT
421 CTGGGCGCCACCGAGGACCGCATCTGCGGCACCATCGACATCGAGAAGGCGCTGACGCAG
481 GGCATCAAGGCCTACGAGCCCGGCCTGCTGGCCAAGGCCAACCGCGGCATCCTGTATGTG
541 GACGAGGTGAACCTGCTGGATGATGGCCTGGTTGATGTCGTGCTGGACTCGTCGGCTAGC
601 GGCCTGAACACTGTGGAGCGTGAGGGTGTGTCCATTGTGCACCCTGCCCGCTTCATCATG
661 ATTGGCTCAGGCAACCCCCAGGAGGGTGAGCTGCGCCCGCAGCTGCTGGATCGCTTCGGC
721 ATGAGCGTCAACGTGGCCACGCTGCAGGACACCAAGCAGCGCACGCAGCTGGTGCTGGAC
781 CGGCTTGCGTACGAGGCGGACCCTGACGCATTTGTGGACTCGTGCAAGGCCGAGCAGACG
841 GCGCTCACGGACAAGCTGGAGGCGGCCCGCCAGCGCCTGCGGTCCGTCAAGATCAGCGAG
901 GAGCTGCAGATCCTGATCTCGGACATTTGCTCGCGCCTGGATGTGGATGGCCTGCGCGGT
961 GACATTGTGATCAACCGCGCCGCCAAGGCGCTTGTGGCCTTCGAGGGCCGCACCGAGGTG
1021 ACCACGAATGACGTGGAGCGCGTCATCTCGGGCTGCCTCAACCACCGCCTGCGCAAGGAC
1081 CCGCTGGACCCCATTGACAACGGCACCAAGGTGGCCATCCTGTTCAAGCGCATGACCGAC
1141 CCCGAGATCATGAAGCGCGAGGAGGAGGCCAAGAAGAAGCGCGAGGAGGCGGCCGCCAAG
1201 GCCAAGGCGGAGGGCAAGGCGGACCGCCCCACGGGCGCCAAGGCTGGCGCCTGGGCTGGC
1261 TTGCCCCCTCGTCGGTAAAAAAGGCTGGTGTAGGCCTGTCGGGTCGTGTTAAAGGTTGCT
1321 GCGTGAACGTGTAAGTGTGACAGTGTGCCGGTATGTGTGTGTATACATGTGTTGCGGTGT
1381 GCTTTTGTGGCGGTACATGGTGATGACTGAGCGGGTGGGACAGAGCACGGTTAACTGACG
1441 AGG
Amino acid sequence
10 20 30 40 50 60
MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDHPFTLPST KAAKKPNFPF VKIQGQEEMK
70 80 90 100 110 120
LALLLNVVDP NIGGVLIMGD RGTAKSVAVR ALVDMLPDID VVEGDAFNSS PTDPKFMGPD
130 140 150 160 170 180
TLQRFRNGEK LPTVRMRTPL VELPLGATED RICGTIDIEK ALTQGIKAYE PGLLAKANRG
190 200 210 220 230 240
ILYVDEVNLL DDGLVDVVLD SSASGLNTVE REGVSIVHPA RFIMIGSGNP QEGELRPQLL
250 260 270 280 290 300
DRFGMSVNVA TLQDTKQRTQ LVLDRLAYEA DPDAFVDSCK AEQTALTDKL EAARQRLRSV
310 320 330 340 350 360
KISEELQILI SDICSRLDVD GLRGDIVINR AAKALVAFEG RTEVTTNDVE RVISGCLNHR
370 380 390 400 410 420
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