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Team:UC-Santa Cruz/Project/Pumps
From 2013.igem.org
Cloning of a Newly discovered Na pumping Halorhodopsin from marine flavobacterium Krokinobacter eikastus
We used a Gibson reation with IDT G Blocks and IGEM pSB1C3 to clone KR2 halorhodopsin into pSB1C3 BioBrick backbone.
Key Reference- A light-driven sodium ion pump in marine bacteria. Inoue et al. Nature Communications DOI: 10.1038/ncomms2689 April 9, 2013.
DNA Sequence KR2 Na pumping halorhodopsin-
ATGACACAAGAACTAGGGAATGCCAATTTCGAAAATTTCATTGGAGCTACAG
AAGGATTTTCTGAAATTGCTTATCAATTTACATCACATATCCTTACGTTAGGGTACGCAGTGATGCTTGCAGGATT
ACTATACTTTATCCTTACCATCAAAAATGTAGATAAAAAATTCCAAATGTCGAACATATTATCAGCTGTGGTAATG
GTATCGGCATTTTTGCTATTATATGCGCAGGCACAAAACTGGACATCCAGTTTTACCTTTAATGAAGAAGTAGGAA
GATATTTTTTAGATCCGAGTGGTGATCTATTTAATAACGGATATCGCTATCTTAACTGGCTCATCGATGTACCTAT
GCTTCTCTTTCAAATTCTATTTGTAGTAAGTTTAACTACTTCAAAATTTAGCTCTGTACGTAACCAATTCTGGTTT
TCTGGGGCAATGATGATTATTACTGGGTACATTGGACAGTTTTATGAGGTAAGTAACTTGACTGCCTTTTTAGTAT
GGGGAGCTATTTCATCTGCTTTTTTCTTCCATATTTTATGGGTTATGAAGAAGGTAATTAATGAAGGAAAAGAGGG
GATTTCCCCAGCAGGACAAAAAATACTTTCTAATATCTGGATCTTATTTTTAATATCATGGACTTTATATCCAGGA
GCTTACTTAATGCCATACCTTACTGGAGTAGACGGATTTTTATATAGTGAAGATGGCGTGATGGCTAGACAACTAG
TATACACTATTGCAGATGTAAGTTCTAAAGTTATCTATGGTGTATTATTAGGTAACCTAGCAATTACATTAAGTAA
AAACAAAGAGTTGGTTGAAGCAAATAGCTAA
Protein Sequence KR2 Na pumping halorhodopsin-
MTQELGNANFENFIGATEGFSEIAYQFTSHILTLGYAVMLAGLLYFILTIKN
VDKKFQMSNILSAVVMVSAFLLLYAQAQNWTSSFTFNEEVGRYFLDPSGDLFNNGYRYLNWLIDVPMLLFQILFVV
SLTTSKFSSVRNQFWFSGAMMIITGYIGQFYEVSNLTAFLVWGAISSAFFFHILWVMKKVINEGKEGISPAGQKIL
SNIWILFLISWTLYPGAYLMPYLTGVDGFLYSEDGVMARQLVYTIADVSSKVIYGVLLGNLAITLSKNKELVEANS*
Length: 280
Molecular weight: 31.524 kDa
Isoelectric point: 4.96
The codon
Usage table for Caulobacter crescentus-
|
Amino Acid |
Codon |
Number/ Genome |
#/1000 |
Fraction |
|
Ala |
GCG |
57020 |
46.06 |
0.334895631 |
|
Ala |
GCA |
3967 |
3.2 |
0.023299386 |
|
Ala |
GCT |
10175 |
8.22 |
0.059760839 |
|
Ala |
GCC |
99100 |
80.04 |
0.582044144 |
|
Arg |
AGG |
2701 |
2.18 |
0.029839039 |
|
Arg |
AGA |
1059 |
0.86 |
0.011699201 |
|
Arg |
CGG |
19055 |
15.39 |
0.210508291 |
|
Arg |
CGA |
4608 |
3.72 |
0.05090644 |
|
Arg |
CGT |
10018 |
8.09 |
0.110672897 |
|
Arg |
CGC |
53078 |
42.87 |
0.586374131 |
|
Asn |
AAT |
5773 |
4.66 |
0.196547733 |
|
Asn |
AAC |
23599 |
19.06 |
0.803452267 |
|
Asp |
GAT |
16529 |
13.35 |
0.231087561 |
|
Asp |
GAC |
54998 |
44.42 |
0.768912439 |
|
Cys |
TGT |
1309 |
1.06 |
0.142732526 |
|
Cys |
TGC |
7862 |
6.35 |
0.857267474 |
|
End |
TGA |
1799 |
1.45 |
0.472550565 |
|
End |
TAG |
1247 |
1.01 |
0.327554505 |
|
End |
TAA |
761 |
0.61 |
0.19989493 |
|
Gln |
CAG |
34155 |
27.59 |
0.859829318 |
|
Gln |
CAA |
5568 |
4.5 |
0.140170682 |
|
Glu |
GAG |
47647 |
38.48 |
0.713289121 |
|
Glu |
GAA |
19152 |
15.47 |
0.286710879 |
|
Gly |
GGG |
13983 |
11.29 |
0.126510929 |
|
Gly |
GGA |
5358 |
4.33 |
0.048476404 |
|
Gly |
GGT |
11281 |
9.11 |
0.102064635 |
|
Gly |
GGC |
79906 |
64.54 |
0.722948031 |
|
His |
CAT |
6487 |
5.24 |
0.293343583 |
|
His |
CAC |
15627 |
12.62 |
0.706656417 |
|
Ile |
ATA |
790 |
0.64 |
0.014615555 |
|
Ile |
ATT |
3549 |
2.87 |
0.065658995 |
|
Ile |
ATC |
49713 |
40.15 |
0.91972545 |
|
Leu |
TTG |
8478 |
6.85 |
0.068407378 |
|
Leu |
TTA |
427 |
0.34 |
0.003445382 |
|
Leu |
CTG |
84529 |
68.27 |
0.68204851 |
|
Leu |
CTA |
1742 |
1.41 |
0.014055868 |
|
Leu |
CTT |
7698 |
6.22 |
0.062113706 |
|
Leu |
CTC |
21060 |
17.01 |
0.169929156 |
|
Lys |
AAG |
40138 |
32.42 |
0.924284991 |
|
Lys |
AAA |
3288 |
2.66 |
0.075715009 |
|
Met |
ATG |
26760 |
21.61 |
1 |
|
Phe |
TTT |
5212 |
4.21 |
0.119489213 |
|
Phe |
TTC |
38407 |
31.02 |
0.880510787 |
|
Pro |
CCG |
37265 |
30.1 |
0.552000474 |
|
Pro |
CCA |
2760 |
2.23 |
0.040883438 |
|
Pro |
CCT |
4338 |
3.5 |
0.064258099 |
|
Pro |
CCC |
23146 |
18.7 |
0.342857989 |
|
Ser |
AGT |
1687 |
1.36 |
0.026704447 |
|
Ser |
AGC |
20348 |
16.44 |
0.322099631 |
|
Ser |
TCG |
26206 |
21.17 |
0.41482912 |
|
Ser |
TCA |
2127 |
1.72 |
0.033669447 |
|
Ser |
TCT |
2144 |
1.73 |
0.03393855 |
|
Ser |
TCC |
10661 |
8.61 |
0.168758805 |
|
Thr |
ACG |
20611 |
16.65 |
0.318075896 |
|
Thr |
ACA |
1775 |
1.43 |
0.027392398 |
|
Thr |
ACT |
1768 |
1.43 |
0.027284372 |
|
Thr |
ACC |
40645 |
32.83 |
0.627247334 |
|
Trp |
TGG |
17312 |
13.98 |
1 |
|
Tyr |
TAT |
11882 |
9.6 |
0.459918715 |
|
Tyr |
TAC |
13953 |
11.27 |
0.540081285 |
|
Val |
GTG |
37323 |
30.15 |
0.397755611 |
|
Val |
GTA |
1517 |
1.23 |
0.016166848 |
|
Val |
GTT |
6714 |
5.42 |
0.07155189 |
|
Val |
GTC |
48280 |
39 |
0.514525652 |
Codon optimized sequence for KR2 Na pumping halorhodopsin
ATGACGCAAGAACTGGGGAATGCCAATTTCGAAAATTTCATCGGTGCGACCG
AAGGCTTTAGCGAAATCGCCTATCAATTTACGTCGCATATCCTCACGCTGGGGTACGCGGTGATGCTCGCCGGGCT
GCTCTACTTTATCCTGACCATCAAGAATGTCGATAAGAAGTTCCAAATGTCGAACATCCTCAGCGCGGTGGTCATG
GTGTCGGCCTTTCTGCTCCTGTATGCGCAGGCGCAAAACTGGACCTCCTCGTTTACCTTTAATGAAGAAGTCGGTC
GTTATTTTCTCGATCCGAGCGGTGATCTGTTTAATAACGGCTATCGCTATCTCAACTGGCTCATCGATGTGCCCAT
GCTGCTCTTTCAAATCCTGTTTGTCGTGTCGCTCACGACCAGCAAGTTTAGCTCGGTCCGTAACCAATTCTGGTTT
AGCGGGGCCATGATGATCATCACGGGGTACATCGGGCAGTTTTATGAGGTGTCGAACCTGACCGCCTTTCTCGTCT
GGGGTGCGATCAGCTCGGCCTTTTTCTTCCATATCCTGTGGGTGATGAAGAAGGTCATCAATGAAGGCAAGGAGGG
GATCTCCCCGGCGGGGCAAAAGATCCTCAGCAATATCTGGATCCTGTTTCTCATCTCGTGGACGCTGTATCCCGGT
GCCTACCTCATGCCGTACCTGACCGGCGTGGACGGGTTTCTCTATAGCGAAGATGGCGTGATGGCGCGCCAACTGG
TCTACACGATCGCCGATGTGTCGAGCAAGGTCATCTATGGTGTGCTCCTGGGTAACCTCGCGATCACCCTGTCGAA
GAACAAGGAGCTCGTCGAAGCCAATAGCTAA
Protein Sequence for KR2 Na
pumping halorhodopsin-
MTQELGNANFENFIGATEGFSEIAYQFTSHILTLGYAVMLAGLLYFILTIKN
VDKKFQMSNILSAVVMVSAFLLLYAQAQNWTSSFTFNEEVGRYFLDPSGDLFNNGYRYLNWLIDVPMLLFQILFVV
SLTTSKFSSVRNQFWFSGAMMIITGYIGQFYEVSNLTAFLVWGAISSAFFFHILWVMKKVINEGKEGISPAGQKIL
SNIWILFLISWTLYPGAYLMPYLTGVDGFLYSEDGVMARQLVYTIADVSSKVIYGVLLGNLAITLSKNKELVEANS*
Alignment of two protein sequences,
one wild type and one codon optimized for Caulobacter (to confirm we have the same protein)
MTQELGNANFENFIGATEGFSEIAYQFTSHILTLGYAVMLAGLLYFILTIKN
VDKKFQMSNILSAVVMVSAFLLLYAQAQNWTSSFTFNEEVGRYFLDPSGDLFNNGYRYLNWLIDVPMLLFQILFVV
SLTTSKFSSVRNQFWFSGAMMIITGYIGQFYEVSNLTAFLVWGAISSAFFFHILWVMKKVINEGKEGISPAGQKIL
SNIWILFLISWTLYPGAYLMPYLTGVDGFLYSEDGVMARQLVYTIADVSSKVIYGVLLGNLAITLSKNKELVEANS
*
MTQELGNANFENFIGATEGFSEIAYQFTSHILTLGYAVMLAGLLYFILTIKN
VDKKFQMSNILSAVVMVSAFLLLYAQAQNWTSSFTFNEEVGRYFLDPSGDLFNNGYRYLNWLIDVPMLLFQILFVV
SLTTSKFSSVRNQFWFSGAMMIITGYIGQFYEVSNLTAFLVWGAISSAFFFHILWVMKKVINEGKEGISPAGQKIL
SNIWILFLISWTLYPGAYLMPYLTGVDGFLYSEDGVMARQLVYTIADVSSKVIYGVLLGNLAITLSKNKELVEANS
*
BioBrick pSB1C3 sequence with KR2 inserted- 2913 bp.
TACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCC
TGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGG
TCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGC
AGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAC
AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA
GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTC
CGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTT
CGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG
AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGT
AGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCT
CTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTG
GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG
GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAG
ATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGGC
TTGGATTCTCACCAATAAAAAACGCCCGGCGGCAACCGAGCGTTCTGAACAAATCCAGATGGAGTTCTGAGGTCAT
TACTGGATCTATCAACAGGAGTCCAAGCGAGCTCGATATCAAATTACGCCCCGCCCTGCCACTCATCGCAGTACTG
TTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATC
AGCACCTTGTCGCCTTGCGTATAATATTTGCCCATGGTGAAAACGGGGGCGAAGAAGTTGTCCATATTGGCCACGT
TTAAATCAAAACTGGTGAAACTCACCCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGGAA
ATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGTGTAGAAACTGCCGGAAATCGTCGTGGTAT
TCACTCCAGAGCGATGAAAACGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCCCATATCA
CCAGCTCACCGTCTTTCATTGCCATACGAAATTCCGGATGAGCATTCATCAGGCGGGCAAGAATGTGAATAAAGGC
CGGATAAAACTTGTGCTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACGGTCTGGTTATAG
GTACATTGAGCAACTGACTGAAATGCCTCAAAATGTTCTTTACGATGCCATTGGGATATATCAACGGTGGTATATC
CAGTGATTTTTTTCTCCATTTTAGCTTCCTTAGCTCCTGAAAATCTCGATAACTCAAAAAATACGCCCGGTAGTGA
TCTTATTTCATTATGGTGAAAGTTGGAACCTCTTACGTGCCCGATCAACTCGAGTGCCACCTGACGTCTAAGAAAC
CATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCT
TTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAG/ATGACGCAAGAACTGGGGAATGCCAATTTCGAA
AATTTCATCGGTGCGACCGAAGGCTTTAGCGAAATCGCCTATCAATTTACGTCGCATATCCTCACGCTGGGGTACG
CGGTGATGCTCGCCGGGCTGCTCTACTTTATCCTGACCATCAAGAATGTCGATAAGAAGTTCCAAATGTCGAACAT
CCTCAGCGCGGTGGTCATGGTGTCGGCCTTTCTGCTCCTGTATGCGCAGGCGCAAAACTGGACCTCCTCGTTTACC
TTTAATGAAGAAGTCGGTCGTTATTTTCTCGATCCGAGCGGTGATCTGTTTAATAACGGCTATCGCTATCTCAACT
GGCTCATCGATGTGCCCATGCTGCTCTTTCAAATCCTGTTTGTCGTGTCGCTCACGACCAGCAAGTTTAGCTCGGT
CCGTAACCAATTCTGGTTTAGCGGGGCCATGATGATCATCACGGGGTACATCGGGCAGTTTTATGAGGTGTCGAAC
CTGACCGCCTTTCTCGTCTGGGGTGCGATCAGCTCGGCCTTTTTCTTCCATATCCTGTGGGTGATGAAGAAGGTCA
TCAATGAAGGCAAGGAGGGGATCTCCCCGGCGGGGCAAAAGATCCTCAGCAATATCTGGATCCTGTTTCTCATCTCGTGGACGCTGTATCCCGGTGCCTACCTCATGCCGTACCTGACCGGCGTGGACGGGTTTCTCTATAGCGAAGA
TGGCGTGATGGCGCGCCAACTGGTCTACACGATCGCCGATGTGTCGAGCAAGGTCATCTATGGTGTGCTCCTGGGT
AACCTCGCGATCACCCTGTCGAAGAACAAGGAGCTCGTCGAAGCCAATAGCTAA/
