I. PROMOTORES
De plantas
http://www.softberry.com/berry.phtml?topic=plantprom&group=data&subgroup=plantprom
http://plantpan2.itps.ncku.edu.tw/sequence.php
Promotor Actina ArrozTCGAGGTCATTCATATGCTTGAGAAGAGAGTCGGGATAGTCCAAAATAAAACAAAGGTAAGATTACCTGG
TCAAAAGTGAAAACATCAGTTAAAAGGTGGTATAAAGTAAAATATCGGTAATAAAAGGTGGCCCAAAGTGAAATT
TACTCTTTTCTACTATTATAAAAATTGAGGATGTTTTTGTCGGTACTTTGATACGTCATTTTTGTATGAATTGGT
TTTTAAGTTTATTCGCTTTTGGAAATGCATATCTGTATTTGAGTCGGGTTTTAAGTTCGTTTGCTTTTGTAAATA
CAGAGGGATTTGTATAAGAAATATCTTTAGAAAAACCCATATGCTAATTTGACATAATTTTTGAGAAAAATATAT
ATTCAGGCGAATTCTCACAATGAACAATAATAAGATTAAAATAGCTTTCCCCCGTTGCAGCGCATGGGTATTTTT
TCTAGTAAAAATAAAAGATAAACTTAGACTCAAAACATTTACAAAAACAACCCCTAAAGTTCCTAAAGCCCAAAG
TGCTATCCACGATCCATAGCAAGCCCAGCCCAACCCAACCCAACCCAACCCACCCCAGTCCAGCCAACTGGACAA
TAGTCTCCACACCCCCCCACTATCACCGTGAGTTGTCCGCACGCACCGCACGTCTCGCAGCCAAAAAAAAAAAGA
AAGAAAAAAAAGAAAAAGAAAAAACAGCAGGTGGGTCCGGGTCGTGGGGGCCGGAAACGCGAGGAGGATCGCGAG
CCAGCGACGAGGCCGGCCCTCCCTCCGCTTCCAAAGAAACGCCCCCCATCGCCACTATATACATACCCCCCCCTC
TCCTCCCATCCCCCCAACCCT
Promoter from cauliflower mosaic virus CaMV 35S
TGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTG
AAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCT
CTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTC
TTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGAC
CCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACA
P-ract +ract1 intron; 5' region of rice (Oryzae sativa) actin 1 gene containing the promoter, transcription start site and first intron"
CTCGAGGTCATTCATATGCTTGAGAAGAGAGTCGGGATAGTCCAAAATAAAACAAAGGTAAGATTACCTG
GTCAAAAGTGAAAACATCAGTTAAAAGGTGGTATAAAGTAAAATATCGGTAATAAAAGGTGGCCCAAAGT
GAAATTTACTCTTTTCTACTATTATAAAAATTGAGGATGTTTTTGTCGGTACTTTGATACGTCATTTTTG
TATGAATTGGTTTTTAAGTTTATTCGCTTTTGGAAATGCATATCTGTATTTGAGTCGGGTTTTAAGTTCG
TTTGCTTTTGTAAATACAGAGGGATTTGTATAAGAAATATCTTTAGAAAAACCCATATGCTAATTTGACA
TAATTTTTGAGAAAAATATATATTCAGGCGAATTCTCACAATGAACAATAATAAGATTAAAATAGCTTTC
CCCCGTTGCAGCGCATGGGTATTTTTTCTAGTAAAAATAAAAGATAAACTTAGACTCAAAACATTTACAA
AAACAACCCCTAAAGTTCCTAAAGCCCAAAGTGCTATCCACGATCCATAGCAAGCCCAGCCCAACCCAAC
CCAACCCAACCCACCCCAGTCCAGCCAACTGGACAATAGTCTCCACACCCCCCCACTATCACCGTGAGTT
GTCCGCACGCACCGCACGTCTCGCAGCCAAAAAAAAAAAGAAAGAAAAAAAAGAAAAAGAAAAAACAGCA
GGTGGGTCCGGGTCGTGGGGGCCGGAAACGCGAGGAGGATCGCGAGCCAGCGACGAGGCCGGCCCTCCCT
CCGCTTCCAAAGAAACGCCCCCCATCGCCACTATATACATACCCCCCCCTCTCCTCCCATCCCCCCAACC
CTACCACCACCACCACCACCACCTCCACCTCCTCCCCCCTCGCTGCCGGACGACGAGCTCCTCCCCCCTC
CCCCTTCGCCGCCGCCGCGCCGGTAACCACCCCGCCCCTCTCCTCTTTCTTTCTCCGTTTTTTTTTCCGT
CTCGGTCTCGATCTTTGGCCTTGGTAGTTTGGGTGGGCGAGAGGCGGCTTCGTGCGCGCCCAGATCGGTG
CGCGGGAGGGGCGGGATCTCGCGGCTGGGGCTCTCGCCGGCGTGGATCCGGCCCGGATCTCGCGGGGAAT
GGGGCTCTCGGATGTAGATCTGCGATCCGCCGTTGTTGGGGGAGATGATGGGGGGTTTAAAATTTCCGCC
GTGCTAAACAAGATCAGGAAGAGGGGAAAAGGGCACTATGGTTTATATTTTTATATATTTCTGCTGCTTC
GTCAGGCTTAGATGTGCTAGATCTTTCTTTCTTCTTTTTGTGGGTAGAATTTGAATCCCTCAGCATTGTT
CATCGGTAGTTTTTCTTTTCATGATTTGTGACAAATGCAGCCTCGTGCGGAGCTTTTTTGTA
maize polyubiquitin gene promoter
CTGCAGTGCAGCGTGACCCGGTCGTGCCCCTCTCTAGAGATAATGAGCATTGCATGTCTAAGTTATAAAAAAT
TACCACATATTTTTTTTGTCACACTTGTTTGAAGTGCAGTTTATCTATCTTTATACATATATTTAAACTTTAC
TCTACGAATAATATAATCTATAGTACTACAATAATATCAGTGTTTTAGAGAATCATATAAATGAACAGTTAGA
CATGGTCTAAAGGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTATCTTTTTAGTGTGCATGTGTTC
TCCTTTTTTTTTGCAAATAGCTTCACCTATATAATACTTCATCCATTTTATTAGTACATCCATTTAGGGTTTA
GGGTTAATGGTTTTTATAGACTAATTTTTTTAGTACATCTATTTTATTCTATTTTAGCCTCTAAATTAAGAAAA
CTAAAACTCTATTTTAGTTTTTTTATTTAATAATTTAGATATAAAATAGAATAAAATAAAGTGACTAAAAATTA
AACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTTGTTTCGAGTAGATAATGCCAGCCTGT
TAAACGCCGTCGACGAGTCTAACGGACACCAACCAGCGAACCAGCAGCGTCGCGTCGGGCCAAGCGAAGCAGAC
GGCACGGCATCTCTGTCGCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTC
GGCATCCAGAAATTGCGTGGCGGAGCGGCAGACGTGAGCCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGC
ACGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCCGTAATAAATAGACAC
CCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACACACAACCAGATCTCCCCCAAATC
CACCCGTCGGCACCTCCGCTTCAAGGTACGCCGCTCGTCCTCCCCCCCCCCCCCTCTCTACCTTCTCTAGATCG
GCGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTTGTGTTAG
ATCCGTGCTGCTAGCGTTCGTACACGGATGCGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGT
TTCTCTTTGGGGAATCCTGGGATGGCTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTGTTTCG
TTGCATAGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTCGGGTCATCTTTTC
ATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGGGCGGTCGTTCTAGATCGGAGTAGAATTCTGTT
TCAAACTACCTGGTGGATTTATTAATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAA
GATGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGGGTTTTACTGATGCATATACAGAGAT
GCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGTGGTTGGGCGGTCGTTCATTCGTTCTAGATCGGAGTAGAAT
ACTGTTTCAAACTACCTGGTGTATTTATTAATTTTGGAACTGTATGTGTGTGTCATACATCTTCATAGTTACGA
GTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGTGGGTTTTACTGATGCATATACATG
ATGGCATATGCAGCATCTATTCATATGCTCTAACCTTGAGTACCTATCTATTATAATAAACAAGTATGTTTTAT
AATTATTTTGATCTTGATATACTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTT
CATACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTGGTGTTACTTCTGCAG
nopaline synthase terminator and poly(A) signal
GATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATA
ATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTA
TGATTAGAGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTA
TCGCGCGCGGTGTCATCTATGTTACTAGATC
Bacillus
Partes para Bacillus
http://parts.igem.org/Bacillus_subtilis#Plasmid_backbones
Partes de Bacillus subtilis (gram positivo)
http://parts.igem.org/Bacillus_subtilis
E.coli
Promotores
http://parts.igem.org/Promoters/Catalog
Promotores evaluados por Prof. Anderson
http://parts.igem.org/Promoters/Catalog/Anderson
RBS
http://parts.igem.org/Part:BBa_B0034
RBS evaluados por Prof. Anderson
http://parts.igem.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Anderson
Promotor que se activa con tetraciclina
http://parts.igem.org/Part:BBa_R0040
Sequence for pTet inverting regulator. Promoter is constitutively ON and repressed by TetR. TetR repression is inhibited by the addition of tetracycline or its analog, aTc.
Secreción en E.coli https://www.dna20.com/eCommerce/catalog/datasheet/257
LEVADURA
Promotor constitutivo de Levadura
http://partsregistry.org/Part:BBa_K517001
https://doi.org/10.1016/0378-1119(95)00037-7
Expresión en Levadura https://www.dna20.com/eCommerce/catalog/datasheet/200
Secreción https://www.dna20.com/eCommerce/catalog/datasheet/386
>BBa_K2294007 Part-only sequence (160 bp) cggattagaagccgccgagcgggcgacagccctccgacggatgactctcctccgttaattaacttgtaatattctaatcaagcttataaaagagcactgt tgggcgtgagtggaggcgccggaaaaaagcatcgaaaaaagtctccatggtggcggcggg
II. KOZAK
In eukaryotes, targeting the small ribosomal subunit to the mRNA transcript is complicated through the addition of required protein: protein interactions [3]. However, characterization of nucleotide sequences surrounding known translational start sites revealed a DNA consensus sequence (5′-GCCRCCATGG-3′) suggesting a conserved role in translational efficiency [4]. This consensus sequence, now known as the Kozak consensus sequence, has become increasingly valuable as a tool for improving artificial expression constructs. Nevertheless, since the initial characterization in 1987, little has been done to functionally characterize the natural sequences in vertebrates. Furthermore, although the Kozak consensus sequence has been incorporated in a multitude of expression studies, no in vivo validation has been done to correlate the Kozak sequence with functional expression levels. (In this manuscript, we utilize the term “Kozak sequence” to refer to the sequence encoding the translation initiation site, and the term “canonical Kozak sequence” to refer to the original consensus sequence described by Kozak in 1987).
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108475
Kozak compiled 211 genes (4) and 699 genes (5) primarily from vertebrates and obtained the above sequence. This sequence was initially thought to be essential for all eukaryotes (4). Later, however, it was revealed that a preferred nucleotide sequence around the initiation codon varies considerably among different species.
The preferred sequences are
GCGGC(A/C)(A/G)(A/C)CAUGGCG for Monocots (1127 genes), AAAAAAA(A/C)AAUGGCU for Dicots (derived from 3643 genes) (6), ACAACCAAAAUGGC for Drosophila melanogaster (192 genes), UAAAT(A/C)AACAUG(A/G)C for other invertebrates (155 genes), and AAAAAAAAAAUGTC for Saccharomyces cerevisiae (461 genes) (3)
Kozak also reported that replacement of A/G at position −3 (three bases before the initiation codon) and G at position +4 (one base after the initiation codon) strongly impaired translation initiation in mammals (7,8). However, in S. cerevisiae nucleotide substitutions at position −3 did not substantially affect the rate of translation initiation (9,10), although there is a nucleotide bias towards A at this position (3). It therefore appears that the molecular mechanisms for recognizing the initiation codon vary among species.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2241899/
http://eprints.soton.ac.uk/397956/1.hasCoversheetVersion/James%20SchofieldPhDFinalThesis.pdf
II. PEPTIDOS SEÑAL
Péptidos señal en E.coli para Secreción
1 PelB MKYLLPTAAAGLLLLAAQPAMA AAA24848
2 hGH MATGSRTSLLLAFGLLCLPWLQEGSA AAA72555
3 PhoA MKQSTIALALLPLLFTPVTKA P00634
4 PhoE MKKSTLALVVMGIVASASVQA P02932
5 OmpA MKKTAIAIAVALAGFATVAQA P0A910
6 OmpF MMKRNILAVIVPALLVAGTANA P02931
7 OmpT MRAKLLGIVLTTPIAISSFA P09169
8 OmpC MKVKVLSLLVPALLVAGAANA P06996
9 LPP MKATKLVLGAVILGSTLLAG P69776
10 STII MKKNIAFLLASMFVFSIATNAYA P22542
11 ST-IA/ST-P MKKLMLAIFISVLSFPSFS P01559
12 LTB MNKVKCYVLFTALLSSLYAHG P13811
13 LamB MMITLRKLPLAVAVAAGVMSAQAMA P02943
14 MalE MKIKTGARILALSALTTMMFSASALA P0AEX9
15 Endo-1,4-beta-xylanase MFKFKKKFLVGLTAAFMSISMFSATASA Q59256
16 DsbA MKKIWLALAGLVLAFSASA P0AEG4
17 L-Asparaginase II MEFFKKTALAALVMGFSGAALA P00805
18 MglB MNKKVLTLSAVMASMLFGAAAHA P0AEE5
19 TorT MRVLLFLLLSLFMLPAFS P38683
20 TolB MKQALRVAFGFLILWASVLHA P0A855
21 sfmC MMTKIKLLMLIIFYLIISASAHA P77249
22 bla MSIQHFRVALIPFFAAFCLPVFA P62593
23 npr MGLGKKLSVAVAASFMSLTISLPGVQA P06832
Ejemplos de secreción. https://slideplayer.com/slide/8065103/
Secreción de Gram positivos
The following list of signal peptides gives an overview of all signal peptides which are currently part of the Signal Peptide Mix of the Signal Peptide Toolbox.
Péptidos señal (Eukariotas)
Some popular signal motifs used to target fluorescent proteins, fusion constructs, and genetically encoded sensors to subcellular compartments http://physrev.physiology.org/content/90/3/1103
IV. Linkers
Linkers para proteínas fusionadas
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726540/
Examples of linkers and their functionalities.
aProtease sensitive cleavage sites are indicated with “↓”
bFactor XIa/FVIIa sensitive cleavage
cMatrix metalloprotease-1 sensitive cleavage sequences, one example provided here
dHIV PR (HIV-1 protease); NS3 protease (HCV protease); Factor Xa sensitive cleavage, respectively
eFurin sensitive cleavage
fCathepsin B sensitive cleavage
V.Codon Optimizer
https://www.idtdna.com/CodonOpt
https://www.novoprolabs.com/tools/codon-optimization
http://genomes.urv.es/OPTIMIZER/
VI. Ejemplos
1. Constructos de Saccharomyces cerevisiae
GAL1 promoter from S. cerevisiae Contains Kozak sequence, part BBa_J63003.
CDS
The ADH1 terminator BBa_K1486025
Constructo 1 .
CCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATT
GCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTC
CGTGCGTCCTCGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCG
AACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGC
CCCACAAACCTTCAAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTA
GCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATG
CAAAAACTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAA
ATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGGAAACT
AGACCCGCCGCCACCATGGAG
ATG XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXTAA
GCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAA
TTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTC
AGGTTGCTTTCTCAGGTATAGTATGAGGTCGCTCTTATTGACCACACCTCTACCGG
2. Constructo transformante de Bacillus subtilis
Promotor Pglv
RBS BBa_K090505
CDS
Terminador BBa_B0010
acatgggaaaactgtatgtatttgatcctc
TCTAGAGAAAGANNNGANNNTACTAG
ATGXXXTAA
ccag
gcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgct
ctc
Constructo†transformante†de†Chlamydomonas†reinhardtii†utilizando†un
promotor†hipotético†de†Arabidopsis†thaliana
Promotor HSP70A/RBSC2
Secuencia kosak
CDS
Terminador rbcL
TCGCTGAGGCTTGACATGATTGGTGCGTATGTTTGTATGAAGCTACAGGACTGATTTGGCGGGCTATG
AGGGCGGGGGAAGCTCTGGAAGGGCCGCGATGGGGCGCGCGGCGTCCAGAAGGCGCCATACGGCCCGC
TGGCGGCACCCATCCGGTATAAAAGCCCGCGACCCCGAACGGTGACCTCCACTTTCAGCGACAAACGA
GCACTTATACATACGCGACTATTCTGCCGCTATACATAACCACTCAGCTAGCTTAAGATCCCATCAAG
CTTGCATGCCGGGCGCGCCAGAAGGAGCGCAGCCAAACCAGGATGATGTTTGATGGGGTATTTGAGCA
CTTGCAACCCTTATCCGGAAGCCCCCTGGCCCACAAAGGCTAGGCGCCAATGCAAGCAGTTCGCATGC
AGCCCCTGGAGCGGTGCCCTCCTGATAAACCGGCCAGGGGGCCTATGTTCTTTACCAUGG†
ATGXXXX
XXXXXXXTAA†
CATAAAAAATAAAAACGATGTTTATGCTATTCACATAAACATCATGAAAAATAAAAATT
Constructos†transformante†de†Pseudomonas†putida
Promotor “ BBa_K864400 ”
RBS “ BBa_B0029 ”
CDS (Peptido señal “ LOLB_PSEAE ”, gen LamB y gen accC1)
Terminador “ BBa_B0024 ”
GAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATT†TACTA
G†TTCACACAGGAAACC†TACTAG†
ATGXXX TACTAG†
TCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA
TACTAGAGAGAGAATATAAAAAGCCAGATTATTAATCCGGCTTTTTTATTATTT
Peptide-oligo
https://www.lifetein.com/Peptide-oligonucleotide-conjugate.html