Extracted from genomic DNA of ''S. cerevisiae'' using PCR.
Order the DNA part from synthesis company
The YY1 transcription factor binding sites on the mCYC minimal promoter. All transcription regulation comes from YY1-binding transcription factors.
Made in our lab with parts from Caroline Ajo-Franklin and David Drubin.
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 6 AA linker as spacer (BBa_K1680000). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
Rick Carrick, Ambhi Ganesan, Alex McMillan
This promoter was isolated by taking 500bp upstream of the MFA1 gene of S. cerevisiae. This promoter will be activated when A factor is expressed in an A haploid yeast cell.
This promotor is on the crick strand of the entered sequence.
Polypeptide containing SV40 nuclear localization sequence (SV40 NLS) for nuclear translocation
designed by oligo hybridisation based on plasmid "pGADT7 AD" from Clontech, w/ RFC25-pre and suffix for protein fusions
SV40 nuclear localization sequence (recognized by importin alpha)
Yeast ADH1 promoter + Firefly Luciferase
Amna Sohail & Molly Petrous & Tyler Knox & Mackenzie Reetz
Yeast promoter + luciferase composite part
AarI AD acceptor vector (pRS305, Gal1P, Adh1t)
This is an acceptor vector for AarI cloning, with A and D ends. Cloning into this plasmid will put your parts under the control of a Gal1 promoter (Gal1P) and Adh1 terminator (Adh1t). For more information regarding AarI acceptor vectors, and AarI cloning in general, please visit the UCSF 2008 wiki.
The pRS305 vector has been modified with an AarI cloning cassette inserted between the PspOMI and SacI sites. Please note, the actual orientation of this vector is reversed.
A modified pRS305 plasmid--Integrating yeast expression vector (LEU2 marker).
This is an alpha amylase gene obtained from Bacillus amyloliquefaciens. It is flanked by a Kozak sequence (Part BBa_K165002) and ADH1 terminator (Part BBa_K392003). The construct is contained in the pSB1C3 plasmid.
This came from the published sequence for Bacillus amyloliquefaciens.
This is the full length ADH1 promoter including the distal and proximal portions and their regulatory elements.
There were three options for the length of the promoter - short, middle, and long. The middle length was chosen.
The part was extracted from S. cerevisiae, YPH500 strain, using colony PCR protocols. It consists of 720 bp upstream of ADH1 gene according to SGD sequence.
nuclear 5-aminolevulinate synthase gene from S. cerevisiae, recoded for mitochondrial expression
The first step in S. cerevisiae's pathway for heme biosynthesis is catalyzed by the product of the HEM1 gene [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=HEM1]. HEM1 is a nuclear gene (1.647 kb,[http://db.yeastgenome.org/cgi-bin/getSeq?seq=YDR232W&flankl=0&flankr=0&map=a3map]) that encodes the enzyme 5-aminolevulinate ("ALA" or "d-ALA") synthase (549 aa,[http://db.yeastgenome.org/cgi-bin/getSeq?seq=YDR232W&flankl=0&flankr=0&map=p3map]). The enzyme is post-translationally imported into the mitochondria where it is responsible for converting glycine and succinyl-CoA to 5-aminolevulinate ("d-ALA") plus CoA and CO2. Deletion of HEM1 is lethal for the cell but viabilty can be restored by providing the cells with the product of the HEM1p catalyzed reaction, namely d-ALA. In an effort to directly express the HEM1 gene from the mitochondrial genome, the HEM1 gene was recoded by DNA 2.0 for mitochondrial codons, an epitope tag (HA = YPYDVPDYA) was added to the C-terminal portion of the protein just before the stop codon, and flanking sequences (100 bp upstm of ATG and 92 dwstm of stop) were added to facilitate integration into mitochondrial genome.
codon optimized for mitochondrial gene expression, HA-epitope C term (not N-term where mt import sequences rest), flank coding seq with homology to COX3 gene of mitochondria to allow for integration and disruption of this gene (should make respiration+ cells become respiration-), and finally added BB prefix and suffix to allow cloning into std Registry vectors
direct synthesis from DNA 2.0, summer 2006
Yeast exportable Trp-rich peptide w/enhanced import (1)
This composite device produces and '''exports''' a '''Histidine rich''' peptide, that is '''import enhanced''' (thanks to a trojan peptide). This device is intended to be used with '''Yeast chassis''', so it is composed by subparts designed also to be use in yeast. The high level structure of the device is: * '''''Kozak''''' consensus sequence for initiation of translation ''(from yeast α-factor mating pheromone (MFα1))'' * '''''Signal''''' peptide that targets the product of the gene for secretion ''(also from yeast α-factor mating pheromone (MFα1))'' * '''''Trojan''''' peptide, to increase internalization in target cell ''(from HIV TAT penetratin)'' * '''''Payload''''': this is the exported '''Histidine''' rich domain of the protein ''(a His-Tag)'' The device's sequence has inherited some useful '''restriction sites''' (''BamHI'' & ''HindIII'') from the parts it's made of. '''Important note''': this part has been submitted to the registry using a direct synthesis DNA sample, so it does not contain scars from ''BB assembly standard''.
plasmid from Jansen lab Tuebingen University
Import enhancer - Polyarginine (trojan peptide)
Trojan peptides are short sequences that penetrate through the plasma membrane inside the cell without the need of any receptor or endocitosis process [Derossi 1998]. They can be used to increase the efficiency with which a protein enters a cell. This part is Polyarginine, and has to be used just before the coding sequence of the peptide that you want to be import enhanced. (refer to ''Characterisation of cell-penetrating peptide-mediated peptide delivery'' [Jones et al 2005] for more information about polyarginine)
Sequence obtained by retro-translation. Codon and mRNA-secondary structure optimized for yeast.
Characterisation of cell-penetrating peptide-mediated peptide delivery [Jones et al 2005]
Gli1 bs + MET25+ LexA repressor (CFPx2 tagged) on pRS304*
pRS315 yeast shuttle vector with Biobrick MCS
A Shuttle vector for yeast. The vector is compatible with RFC10 standard and contains an ADH terminator behind the RFC10 cloning site. Ampicilin resistance for bakteria and URA3 gene for yeast selection.
contains an RFC10 compatible multiple cloning site, no restrictions sites for RFC10 in the Backbone. ADH Terminator after MCS.
(+)-Limonene synthase 1 with Strep-tag and yeast consensus sequence.
This part contains the (+)-Limonene Synthase 1 of ''Citrus limon''. It is preceeded by the yeast consensus sequence for improved expression and carries a C-Terminal Strep-Tag for purification or detection by westernblot. It is an improved version of BBa_I742111.
5' UTR site of alcohol oxidase 1 gene (aox1)
5' UTR site of alcohol oxidase 1 gene (AOX1) from K. pastoris used as a recombination site.
(+)-limonene synthase 1 expression cassette for yeast
This part can be used to express Citrus limon (+)-limonene synthase 1 in yeast. The expression is controled by the TEF1 promoter and the CYC1 terminator.
This is a composite part consisting of the yeast TEF1 promoter (BBa_K319003), the ''Citrus limon'' (+)-limonene synthase 1 coding region (BBa_K801061) and the yeast CYC1 terminator (BBa_K122003).
This is the venus YFP, yeast optimized in the Silver Lab BioFusion standard for fusion of protein domains.
This is an alpha amylase promoter derived from Bacillus amyloliquefaciens. It is flanked by Kozak sequence (Part BBa_K165002) the ADH1 terminator (Part BBa_K392003) . Expression is controlled by the cyc yeast promoter (Part BBa_K105027).
The amylase gene was obtained from the published sequence for Bacillus amyloliquefaciens
This is the exact region between the end of the ORF of STE2-W and the beginning of BST1-C
This sequence was taken from the yeast genome
AarI AD acceptor vector (pRS315, Adh1P, Adh1t)
This is an acceptor vector for AarI cloning, with A and D ends. Cloning into this plasmid will put your parts under the control of a Adh1 promoter (Adh1P) and Adh1 terminator (Adh1t). For more information regarding AarI acceptor vectors, and AarI cloning in general, please visit the UCSF 2008 wiki.
The pRS315 vector has been modified with an AarI cloning cassette inserted between the PspOMI and SacI sites.
This is the 1500 bp upstream of the yeast PGK1 coding region. This is a putative constitutive promoter.
Source: Saccharomyces cerevisiae genomic DNA.
This part was obtained from yeast genomic DNA.
hsl1 - cell cycle dependent degradation tag in yeast
This BioBrick is a fragement of the yeast hsl1 protein. Fused to an artificial transcription factor it tiggers the degradation in??? phase via the ???.
To enable the fusion with the protein in frame the vector of this BioBrick has no base pair in between the restriction sites and the BioBrick. Furthermore, this coding sequence does not include a start codon. For more information about this issus, see: Phillips, I.E. and Silver, P.A. "A new Biobrick Assembly Strategy Designed for Facile Protein Engineering." DSpace http://hdl.handle.net/1721.1/32535 (2006).
Gli1 binding sites + MET25 inducible yeast promoter
This is a high level constitutive promoter from the yeast gene TEF2.
From the yeast genome using promoter definition from: Mumberg, Muller, and Funk: http://dx.doi.org/10.1016/0378-1119(95)00037-7
SV40 nuclear localization sequence from SV40; yeast codon optimized
yeast codon-optimized nuclear localization sequence from simian virus 40
direct synthesis from published sequence
This is a derivate of BBa_K105027. Due to a point mutation in the TATA box region the efficiency of this minimal promoter is only 70 % of the natural yeast CYC1 promoter.
This BioBrick contains the TATA-box with one point mutation and most of the 5'UTR (-139 to -36, relative to the translational start) of the natural CYC1 promoter. A Kozak sequence is not included and has thus to be added! Even if there isn't any need for this BioBrick to be fusible to other protein domains, we stayed with our design.
Amplification and Mutation from the plasmid pAH3
Lactate Permease from Kluyveromyces lactis
Ra??ssa Estrela Curado, Ta??s Sineiro Herig and Gleidson Silva Teixeira
JEN1 from Kluyveromyces lactis is a homologue gene to JEN1 of Saccharomyces cerevisiae. It ecodes a monocarboxylate acids permease shared by lactic and pyruvic acids. Queiros O, Pereira L, Paiva S, Moradas-Ferreira P and Casal M. 2007. Functional analysis of Kluyveromyces lactis carboxylic acids permeases: heterologous expression of KlJEN1 and KlJEN2 genes. Curr Genet 51, 161-169.
This part was amplified by PCR from K. lactis genomic DNA.
contains restriction sites needed for assembly of BioBricks
Full LexA Repressor (tagges with mCherryx2)
Kozak+LexA DBD+Sin3p RD+mCherryx2+SV40NLS+tADH1
This is a derivate of BBa_K105027. Due to a point mutation in the TATA box region the efficiency of this minimal promoter is only 16 % of the natural yeast CYC1 promoter.
Odr-10(diacetyl)GPCR with optimized codon
Junqi kuang, Xihao liao and Jianhui gong
Odr-10(diacetyl)GPCR encodes a diacetyl-sensing G-protein coupled receptor, originating from the C. elegans. If BBa_K1072010 was transformed into the yeast, the pheromone receptors will be actived and coupled to a heterotrimeric G protein, Odr-10. pathway is composedof Gpa1p (Gα), Ste4p (Gβ) and Ste18p (Gγ) subunits.The Gβγ particle transmits the signal to a MAP kinase module and finally follow a inducible promotor FUS1, which will link a reproter, such as GFP, His3 or LacZ.
As discription of most paper, RNA of Odr-10 be used as plate and transform into receptor cells to express member protein successfully.
pTUM101 yeast shuttle vector with pTEF1 promoter
This part corresponds to pTUM100 (BBa_K801000). The constitutive pTEF1 promoter has been cloned in front of the MCS (RFC25 prefix and suffix).
All forbidden restriction sites have been removed from the plasmid backbone, making it RFC25 compatible
Based on invitrogen's pYES2 plasmid. For mor information see the part information of pTUM100 (BBa_K801000).
Aga2 CDS Responsible for Yeast Surface Display
The Aga2 protein is fused in frame with a protein that the user wants to display on the yeast cellular surface. Yeast cells constitutively expressing Aga1 (strain EBY100) secrete the Aga2 fusion protein, which associates with Aga1 through two disulfide bonds and quaternary structure to anchor Aga2 to the surface. The fused protein then is displayed on the surface of the yeast cell. The STOP codon at the end of the CDS was removed to allow for N- and C-terminus fusions.
We removed the STOP codon to allow for N- and C-terminal fusions, which have both been proven to occur efficiently.
This parts was biobricked from the pCTCON2 plasmid which was sent to us by Dr. D. K. Wittrup at MIT.
extracted from "pGBKT7" from clontech, with RFC25-pre and suffix for protein fusions
Gli1 bs + TEF2 + LexA repressor (CFPx2 tagged) on pRS304*
Tadashi Nakamura, Shuhei Yasumoto, Takahiro Saka, Saya Kakuda
Engineered 'cyc100 promoter' of Saccharomyces cerevisiae with Kozak sequence attached downstream.
Registry parts BBa_K105027 (promoter) and BBa_J63003 (Kozak).
rox1 is a repressor. It targets the promotor anb1.
Sequence and information is based on the Saccharomyces Genome Database, see http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=rox1
Protein coding region for the DNA recombinase Cre, codon optimized for yeast. The Cre recombinase recognizes two loxP sites (Part: BBa_K1680005) and depending on their orientation cuts out or reverses the DNA sequence between them.
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 12 AA linker as spacer (BBa_K1680002). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
Protein coding region for the Hepatitis C virus (HCV) NS3-4A protease with N- & C-terminal linkers. Intended for combination with 145N Dronpa (Part BBa_K1680006) as described by Zhou et al. 2012. Codon optimized for Yeast.
Autonomously Replicating Sequence Binding Factor 1
The Autonomously Replicating Sequence Binding Factor 1, or ABF1 is a site specific DNA-binding protein that can function in both transcriptional activation and repression in Saccharomyces cerevisiae. In particular, the Alcohol Dehydrogenase 1 (ADH1) promoter gene contains two ABF1 binding sites in its promoter and coding regions. Both ABF1 binding sites have been shown to be transcriptional activators, as deletions and mutations at these sites has decreased transcriptional activity. ABF1 behaves as a transcriptional factor or activator for the ADH1 promoter.
No specific design considerations were dealt with.
Source: NCBI GenBank Organism:Saccharomyces cerevisiae
This is the same part as BBa_K110007 except it has Prefix and Suffix regions switched
This is the whole regulatory region from the URA3 gene coding for OMP decarboxylase in S. cerevisiae budding yeast.
NCBI Sequence database: http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=Nucleotide&dopt=GenBank&val=172061
Import enhancer - HIV TAT penetratin (trojan peptide)
Trojan peptides are short sequences that penetrate through the plasma membrane inside the cell without the need of any receptor or endocitosis process [Derossi 1998]. They can be used to increase the efficiency with which a protein enters a cell. This part is the penetratin from the HIV TAT protein, and has to be used just before the coding sequence of the peptide that you want to be ''import enhanced''.
This promoter was isolated by taking 500bp upstream of the MFA2 gene of S. cerevisiae. This promoter will be activated when A factor is expressed in an A haploid yeast cell.
This gene is on the Watson strand of the entered sequence.
Naringenin - chalcone synthase (CHS) + yeast consensus sequence
Naringenin - chalcone synthase(CHS) coding sequence from ''Hypericum androsaemum'' preceeded by yeast consensus sequence for improved expression.
Plasmid (template for PCR) provided by John A. Morgan, School of Chemical Engineering, Purdue University, West Lafayette, Indiana, USA http://www.ncbi.nlm.nih.gov/nucleotide/11096318/ PMID: 15932991
GAL4 based yeast light-switchable promoter system
SV40 NLS (SV40 nuclear localization signal)
BBa_K801040 (SV40NLS-PhyB-Linker-GAL4DBD)
BBa_K801039 (SV40NLS-GAL4AD-Linker-PIF3)
20 amino acids long linker (BBa_K365005)
GAL4AD (GAL4 transcriptonal activation domain)
Phytochrome B (PhyB, 908 first N-terminal amino acids)
This is the mCherry fluorescent protein, yeast-codon optimized and biobricked in the silver lab BioFusion standard for use in fusion proteins.
3,7-dimethylxanthine N-methyltransferase CaDXMT1-strep
3,7-dimethylxanthine N-methyltransferase CaDXMT1 from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
http://www.ncbi.nlm.nih.gov/nuccore/AB084125 synthesized by IDT, yeast codon optimized
Yeast optimized superfolder GFP C-terminus (215-238)
Zif268-HIV synthetic activator (untagged)
This is a synthetic transcriptional activator for use in yeast. It contains the VP16 activation domain, the Zif268-HIV DNA-binding domain, the SV40 nuclear localization sequence, and the ADH1 terminator. It binds to the Zif268-HIV binding sites, activating transcription downstream.
From David Drubin in the Silver Lab at Harvard.
Yeast ADH1 promoter + Renilla Luciferase
AarI AD acceptor, pRS315, 8X LexAOPs Cyc1P, Adht1
This is an acceptor vector for AarI cloning with 8X Lex operators directly upstream of a Cyc1 promoter. The vector contains the AarI cloning cassette (AD sites, with Adh1 terminator). It is a pRS315 derived vector, for CEN ARS expression in cerevisiae (LEU2 marker).
LexA operator repeats were cloned into the PspOMI site at the 5' end of the Cyc1 promoter. The inner site was broken, thus the operator-promoter-AarI cassette fragment can be subcloned by PspOMI/SacI or Kpn1/SacI digestion into any other pRS series vector.
This is a derivative of part BBa_K106014, the AarI AD acceptor vector (pRS315, Cyc1P, Adh1t).
Yeast expression cassette for CaMXMT1 (BBa_K801071) controlled by the yeast TEF1 promoter (BBa_K319003) and the yeast ADH1 terminator (BBa_K801012).
Composite part of BBa_K319003, BBa_K801071 and BBa_K801012
Gli1 bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS303
This is a optimized firefly luciferase (C-terminal modification, see design notes).
Primers were designed to replace the C-terminal SKL aminoacids with KL.
The original source is the Firefly luciferase, see http://www.ncbi.nlm.nih.gov/nuccore/AB644228.1 The modification is covered by 'One-step measurement of firefly luciferase activity in yeast' (http://www.ncbi.nlm.nih.gov/pubmed/14558144).
SV40NLS-GAL4AD-linker-PIF3 (part for GAL4/LexA based light-switchable promoter system)
CDS for a synthetic hybrid fusion protein. Consists of serveral domains. RFC10 pre and suffix
CDS for a synthetic hybrid fusion protein
Protein fusion vector (Silver lab standard)
standard biofusion plasmid containing ccdB death gene and chlorenphenicol resistance
Stable and soluble Histidine rich peptide.
Retro-translated and optimized (codons and mRNA seconday structure) for yeast.
Prepro-Thaumatin expression cassette for yeast
This part can be used to constitutively express Prepro-Thaumatin in ''S. cerevisiae'' under the control of the TEF1-promoter and the TEF1-terminator.
Based on part BBa_K801080 (Prepro-Thaumatin).
DEC-205 is an antigen against an endocytic receptor that is expressed by dendritic cells. Antigen endocytosed via CD205 enters the MHC class I and MHC class II antigen presentation pathways and is subsequently presented to both CD4+ and CD8+ T cells. We used it to activate T-CD8 cells, with tumorous antigen that we binded to.
Cup-1 is a regulatory promoter that senses heavy metals such as copper, arsenic, cadmium, antimony, mercury, chromium, and selenium.
Comes from genomic DNA of saccharomyces cerevisiae.
Kozak sequence from yeast α-factor mating pheromone (MFα1)
The Kozak sequence is the eukaryotic analog of the bacterial RBS, it is short sequence that includes the ATG, required for proficient initiation of translation. It is known that the 5???UTR sequence of the MFα1 gene of yeast produces efficient initiation of translation. This part is that specific region of the MFα1 gene.
Zif268-HIV bs + MET25+ Gli1 repressor (CFPx2 tagged) on pRS304*
Blue Chromoprotein + 2a + HBsAg + Yeast Secretion Tag + ADH1 Terminator
Brian Yang, Jonathan Krog, Sunil Deochand, Jonathan Kelly
The chimeric GPCR consists of the N- and C- terminals of the rat OR RI7, flanking the TM2-TM7 ligand-binding domain of the c. elegans OR odr-10. It can be functionally expressed in S. cerevisiae, activating the yeast endogenous MAPK pathway upon binding to the ligand diacetyl.
cin8 - cell cycle specific degradation tag in yeast
This BioBrick is the xxx domain of the yeast cin8 protein. It has been shown that this region of part of the protein is suffient to get xxx specific degradation via the APC complex.
To enable the fusion with other domains in frame the vector of this BioBrick has no base pair in between the restriction side and the BioBrick. Furthermore, this coding sequence does not include a start codon.<br\n> For more information about this issus, see:<br\n> Phillips, I.E. and Silver, P.A. "A new Biobrick Assembly Strategy Designed for Facile Protein Engineering." <br\n> DSpace http://hdl.handle.net/1721.1/32535 (2006).
Murine IFN gamma cytokine codon optimized to give the best yield in yeast S. cerevisiae. It promotes T cell activation, upregulates MHC-I and is pro-inflammatory. MAT alpha is the peptide leader used as a secretion signal and fused to IFN gamma without ATG.
We performed a PCR site directed mutagenesis removing an enzyme restriction site in order to make it biobrick compatible. The mutation performed is silencious.
MAT alpha was amplified from the S. cerevisiae genome. IFN gamma was synthesised by IDT based on the genomic murine IFN gamma. Both sequences have been assembled by golden gate to make a single fusion protein.
In the presence of LacI, this sequence represses gene expression. Expression can be induced by the addition of lactose or IPTG, which binds to LacI, removing it from the DNA.
Had to ensure that it would work in yeast and where to put this with regards to the Kozak sequence and protein coding region. Ended up being placed directly before the Kozak sequence.
Was extracted from the BBa_K1164004 part on iGem's Registry of Standard Biological Parts.
Zif268-HIV bs + ADH1 + Gli1 repressor (CFPx2 tagged) on pRS304*
This is a derivate of BBa_K105027. Due to a point mutation in the TATA box region the efficiency of this minimal promoter is only 43 % of the natural yeast CYC1 promoter.
Composite part of BBa_K365005 BBa_K801032
Full Zif268-HIV Repressor (tagged with mCherryx2)
Kozak+Sin3p RD+Zif268-HIV DBD+mCherryx2+SV40NLS+tADH1
Sas2 histone acetyltransferase, Aar1 BD part
This is the gene coding for the Sas2 gene in yeast. It is a histone acetyltransferase (HAT).
Since this is a BD part, there is no ATG but it does include a stop codon. For more information on AarI cloning, please refer to the UCSF 2008 iGEM page.
Yeast CLB1 promoter region, G2/M cell cycle specific
Description of CLB1 (from SGD): B-type cyclin involved in cell cycle progression; activates Cdc28p to promote the transition from G2 to M phase; accumulates during G2 and M, then targeted via a destruction box motif for ubiquitin-mediated degradation by the proteasome. More information found here: http://db.yeastgenome.org/cgi-bin/locus.pl?locus=CLB1
Part contains 500 bases upstream from the CLB1 ORF. Is NOT a minimal promoter, and has not been tested for composability.
Yeast consensus sequence from SGD chrVII:703140..703639
O-methyltransferase 1 (OMT1) coding region
O-methyltransferase 1 (OMT1) coding region from ''Humulus lupulus''
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 9 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
This is the core of the CYC1 promoter of ''S. cerevisiae''. This promoter element allows basal transcription. The transcription signal can be modulated by adding operator sites upstream of this BioBrick. In that way activators or repressors can act on the transcription machinery. <br<
This BioBrick contains the TATA-box and most of the 5'UTR (-139 to -36, relative to the translational start)of the natural CYC1 promoter. A Kozak sequence is not included and has thus to be added!
Tir1 promoter, Saccharomyces cerevisiae cold shock promoter
used to induce the expression of SrUCP under low temperature
cloned from genomic DNA of Saccharomyces cerevisiae
This is a synthetic transcriptional activator for use in yeast. It contains the VP16 activation domain, the LexA DNA-binding domain, the SV40 nuclear localization sequence, and the ADH1 terminator. It binds to the Lex operator, activating transcription downstream.
From David Drubin in Silver Lab at Harvard.
Protein coding region for the firefly [Photinus pyralis] luciferase, codon optimized for yeast.
This secretion tag when linked to the N-terminal of the protein directs extracellular secretion of the protein. After targeting through the ER to the golgi, this "pre-pro" tag is cleaved by the KEX-2 protease resulting in secretion of protein without the aforementioned tag. To use this tag, attached it directly upstream of your proteins CDS.
There was an illegal Xbal1 site at bp 101 and we removed it through altering the primers necessary for biobricking the sequence.
This part was sent to use from Dr. Sheldon Park of the University of Buffalo.
This part confers resistance to Geneticin (G418) in yeast. Selection of yeast cells is typically preformed on plates with 200??g/ml of G418.
The part was synthesized from the PMKV005 plasmid used in McCusker et. al. The coding region contains the KanR open reading from transposon Tn903 from E. coli. 1. McCusker JH, Vorachek-Warren MK. DsdA (D-serine deaminase): a new heterologous MX cassette for gene disruption and selection in Saccharomyces cerevisiae. Yeast (Chichester, England). 2004;21(2):163-71. Available at: http://www.ncbi.nlm.nih.gov/pubmed/14755641.
Fusion protein of TALE2 and a SpyTag in an expression cassette
Coming soon, if I am allowed to edit this.
It keeps saying I have no permission to edit this.
CoxVI is short for the subunit VI of the yeast cytochrome c oxidase, a N-terminal signal peptide to mitochondrial matrix.
CoxVI is obtained from Saccharomyces cerevisiae's genome.
mRFP (DsRed) derived, Ex587/Em610, yeast codon optimized
Codon optimized for yeast. Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2050 (mOrange), BBa_E2020 (Cerulean CFP), and BBa_E2030 (Venus YFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. except for 5' and 3' ends no significant sequence identity runs to mOrange (BBa_E2050).
Shaner et al, 2004. Nat Biotech (22):1567-1571
Mid1 is a voltage gated Calcium channel(VGCC) of yeast which works with Cch1, another VGCC. It was codon optimized for S.cerevisae
With biobrick prefix and suffix added and illegal site eliminated.
enhanced version of EYFP, yeast-optimized
Venus YFP, yeast codon optimized. EX515/EM528
Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2060 (mCherry), BBa_E2050 (mOrange), and BBa_E2020 (Cerulean CFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Inserts a "V" after normal start M. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. Except for 5' and 3' ends no significant sequence identity runs with Cerulean CFP (BBa_E2020) or to GFP S65T as found in O'Shea deletion strain collection (originally from plasmid pFA6-GFP(S65T)-His3MX6).
Yeast ADH1 Promoter + Firefly Luciferase + Yeast ADH1 Promoter + Renilla Luciferase
Yeast Promoter + Luciferase + Yeast Promoter + Renilla
This is the cassette (promoter+coding region+terminator) that imparts resistance to nourseothricin in yeast.
Coding region for Prepro-Thaumatin. Codon optimized for yeast.
Synthesized by IDT. The part is codon optimized for yeast. 3 forbidden restriction sites have been removed.
Based on: http://www.ncbi.nlm.nih.gov/nuccore/121945717
pRS313 yeast shuttle vector with Biobrick MCS
A shuttle vector for yeast. The vector is compatible with RFC10 standard and contains an ADH terminator behind the RFC10 cloning site. Ampicilin resistance for bacteria and URA3 gene for yeast selection.
contains an ADH Terminator and restrictions sites for the RFC10 standard only in the multiple cloning site, no restrictions sites for RCF10 enzymes in the backbone.
This part can be used to express Citrus limon (+)-limonene synthase 1 in yeast. The expression is controled by the yeast TEF1 promoter and the yeast TEF1 terminator.
This is a composite part consisting of the yeast TEF1 promoter (BBa_K319003), the Citrus limon (+)-limonene synthase 1 coding region (BBa_K801061) and the yeast TEF1 terminator (BBa_K801011).
The Gli1 and Zif268-HIV transcription factor binding sites on the mCYC minimal promoter (in that order.) Transcription regulation comes from Gli1 and Zif268-HIV binding transcription factors.
Constructed in our lab with parts from Caroline Ajo-Franklin and David Drubin.
Yeast expression cassette for CaXMT1 (BBa_K801070) controlled by the yeast TEF2 promoter (BBa_K801010) and the yeast ADH1 terminator (BBa_K801012).
Composite part of BBa_K801010, BBa_K801070, BBa_K801012
Gli1 bs + LexA bs + mCYC + LexA repressor (mCherryx2 tagged) on pRS304*
The promoter from the yeast gene CYC1. Activity characterized here: http://dx.doi.org/10.1016/0378-1119(95)00037-7.
From Mumberg, Muller, and Funk p413 pCYC. See ref. above
enhanced version of ECFP, yeast-optimized
Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2060 (mCherry), BBa_E2050 (mOrange), and BBa_E2030 (Venus YFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Inserts a "V" after normal start M. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. Except for 5' and 3' ends no significant sequence identity runs with Venus YFP (BBa_E2030) or to GFP S65T as found in O'Shea deletion strain collection (originally from plasmid pFA6-GFP(S65T)-His3MX6).
Rizzo MA, et al. (2004) Nat Biot (22):445-9
Protein coding region for a mutant (Deletion of AA1-19) of DNA recombinase Cre, codon optimized for yeast.
BBa_K775000 encodes a chimeric niacin-sensing G-protein coupled receptor which can be functionally expressed in S. cerevisiae. This chimeric receptor is composed of the ligand-binding domain of the mammalian GPR109A, flanked by the N- and C- terminals of the rat OR RI7.
rhodopsin siganl peptide +IL-8 receptor type alpha (CXCR1)
Wan-Yun Chiu, Timothy Chan En Haw, Ming-Fei Hsieh
'''TrpZipper''' is a small peptide that folds into a beta-hairpin secondary structure. The indole rings of the Trp form a hydrophobic core. The protein is water soluble and monomer.
Tryptophan zippers: Stable, monomeric beta-hairpins [Cochran 2001]
4-coumarate--coenzyme A ligase (4CL) coding region
4-coumarate--coenzyme A ligase (4CL) coding region from ''Arabidopsis thaliana''.
Plasmid (template for PCR) provided by John A. Morgan, School of Chemical Engineering, Purdue University, West Lafayette, Indiana, USA http://www.ncbi.nlm.nih.gov/nucleotide/609339/ PMID: 15932991
The Gli1 and LexA transcription factor binding sites on the mCYC minimal promoter (in that order.) Transcription regulation comes from Gli1 and LexA-binding transcription factors.
This is the coding region for the Sex-Lethal Gene from which can be used to control splicing events.
This was part was made using PCR from a construct synthesized by Mr. Gene. It intons have been artificially removed.
Kozak + LexA Synthetic Activator (untagged)
This is the kozak ribosome binding sequence ligated onto the LexA synthetic transcriptional activator (BBa_K165020).
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 15 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Stable and soluble '''Histidine''' rich peptide (designed in silico) This part has a '''HindIII''' restriction site as extra.
PolyWb was desing taking into acount the following consideration: # avoided repeating the same residue in tandem to minimize local tRNA depleation # avoided Trp in tandem because of therir low solubility # we included Gly to avoid the formation of stable structures # included acidic and basic amino acids to increase solubility Sequence obtained by retro-translation. Codon and mRNA-secondary structure optimized for yeast.
pGAL1 + Untagged Zif268-HIV activator on pRS305
This is 2 copies of the CFP gene fused to each other. The duplex FP tends to be more stable, and is used in synthetic transcription factors to provide a fluorescent output of concentration as well as stability.
Esa1 histone acetyltransferase, Aar1 BD part
This is the gene sequence for the Esa1 gene with AB ends for Aar1 cloning. Esa1 is a histone acetyltransferase (HAT).
Since this is an AB part, there is a ATG but no stop codon. For more information on AarI cloning, please refer to the UCSF 2008 iGEM page.
Kozak+Sin3p RD+Gli1 DBD+CFPx2+SV40NLS+tADH1
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 15 AA linker as spacer (BBa_K1680003). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
Mid-expression level constitutive promoter in yeast
The is a repressable promoter for use in Saccharomyces cerevisiae. It is repressed by the presence of methionine. When unrepresssed, is has a moderate level of activity.
From the Saccharomyces cerevisiae genome, by way of Caroline Ajo-Franklin.
pTEF2 + Untagged LexA activator on pRS305
AarI A!D acceptor vector (pRS315, Cyc1P, Adh1t)
This is a special variant acceptor vector for AarI cloning. The "A" site used here, termed "A!" is an alternate end used when no amino-terminal fusions are tolerated. Please see the UCSF 2008 iGEM wiki for more information on AarI cloning. This was designed specifically for use with Sir3 and amino-terminal domains of Sir3.
Note that this vector will NOT accept standard AarI "A" parts.
phenylalanine ammonia lyase (PAL) coding region
Coding region for the enzyme phenylalanine ammonia lyase (PAL) from ''Rhodosporidium toruloides''
Plasmid (template for PCR) provided by John A. Morgan, School of Chemical Engineering, Purdue University, West Lafayette, Indiana, USA http://www.ncbi.nlm.nih.gov/nuccore/AX366866 PMID: 15932991
When working correctly this reporter construct transcribes Aga2-eCFP from the Gal promoter. If the yeast strain is also expressing Aga1, then this fusion should be anchored onto the plasma membrane, allowing you to visualize eCFP as a halo around the yeast cell.
We considered fusing this reporter to a variety of promoters, but in the end chose pGAL because it gives a very high level of transcription and we could turn the reporter on and off
This part comes from a mixture of biobricks that were received in the 2010 initial distribution and from parts that NYU iGEM 2010 made ourselves.
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with intermediate 6 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Raissa Estrela Curado, Tais Sineiro Herig and Gleidson Silva Teixeira
This part encodes a reporter cassette for the Cre Recombinase (e.g parts XYZ) in Yeast. An active Cre protein will switch expression from RFP to NanoLuc luciferase.
Promoter of the FIG1 gene in yeast. The transcription factor Ste12, activated in the yeast pheromone pathway, binds to the FIG1 promoter and initiate the expression of the FIG1 gene. Five pheromone response elements (PRE, consensus sequence ATGAAACA) have been identifi?ed in the 5'-upstream regulatory sequence of the FIG1 gene. Phosphorylated Ste12 binds to them and thereby activates transcription. The PREs in the FIG1 promoter di?ffer slightly from the PRE consensus sequence ATGAAACA: Sequence Location TGAAACG -405 TGAAACC* -217 TGACACA* -206 TGAAACG -173 TGAAATA -133 Sites marked with an asterisk occur on the opposite strand The strictly regulated transcriptional feature of the FIG1 promoter can be useful when investigating the pheromone pathway. The promoter can be used to express reporter genes in response to activation of the pheromone pathway. Reference: Erdman S, Lin L, Malczynski M, Snyder M. Pheromone-regulated Genes Required for Yeast Mating Diff?erentiation. The Journal of cell biology. 1998;140(3):461-483.
The length of the promoter was chosen to 450 bp as recommended by Kondo group of Kobe University of Japan. Togawa S, Ishii J, Ishikura A, Tanaka T, Ogino C, Kondo A. Importance of asparagine residues at positions 13 and 26 on the amino-terminal domain of human somatostatin receptor subtype-5 in signalling. J Biochem. 2010 Jun;147(6):867-73
The sequence has been amplified directly from genomic yeast DNA.
LacI promoter with RBS attached downstream.
Registry parts BBa_R0010 (promoter) and BBa_B0034 (RBS).
rLucN + ADH1 terminator + Kanamycin resistance
Zif268-HIV binding sites + MET25 constitutive yeast promoter
pTUM100 yeast shuttle vector based on pYES2
A yeast shuttle vector containing the following structural elements: * 2?? ori for replication in ''S. cerevisiae'' * URA3-gene for selection of transformed yeast via Uracil prototrophy * pUC ori for replication in ''E. coli'' * AmpR-gene for selection of transformed ''E. coli'' via Ampicillin resistance * f1 ori * MCS containing the RFC25 pre- and suffix for easy insertion of biobricks * following the MCS: CYC1 yeast transcriptional terminator Note: To use this backbone, a ''S. cerevisiae'' strain with Uracil-auxotrophy (such as strain INVSc1) is required for positive selection of transformants. Successfully transformed yeast cells will become prototrophic for uracil and can be selected on Agar-plates deficient of uracil.
This is a composite part containing the minimal promoter mCYC preceded by multiple consecutive binding sites for the Gli1 binding domain. This part contains the sequences for BBa_K165012 and BBa_K165016 but also has some additional DNA.
It was necessary to design primers for use in PCR to remove this part from its host plasmid and transfer it to a standard Biobrick vector.
Composite part that was taken via PCR from a synthetic plasmid containing the sequence. The plasmid came from Lawrence Berkeley National Laboratory.
consensus Kozak sequence and start codon
designed such that fusion using fusion bricks assembly method leads to in frame translation
consensus Kozak and start built from oligonucleotides
CoxVI is the N-terminal mitochondrial localization signal from subunit IV of the yeast cytochrome c oxidase.
This part can be used to express ''Citrus limon'' (+)-limonene synthase 1 in yeast. The expression is controled by the yeast TEF2 promoter and the yeast CYC1 terminator.
This is a composite part consisting of the yeast TEF2 promoter (BBa_K801010), the Citrus limon (+)-limonene synthase 1 coding region (BBa_K801061) and the yeast TEF1 terminator (BBa_K122003).
7-methylxanthine N-methyltransferase CaMXMT1-strep
7-methylxanthine N-methyltransferase CaMXMT1 from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
Synthesized by IDT, Yeast codon optimized
TUP1 - repressor domain for transcription factors in yeast
This BioBrick is the N-terminal repression domain of the yeast general repressor TUP1.<br\n> In artificial systems TUP1 is used in fusion with a DNA-binding domain. This DNA-binding domain is specific for a bait sequence. So the selective repressionn of genes with a promoter containing this bait sequence can be achieved.
To enable the fusion with other domains in frame the vector of this BioBrick has no base pair in between the restriction side and the BioBrick.<br\n> For more information about this issus, see Phillips, I.E. and Silver, P.A. "A new Biobrick Assembly Strategy Designed for Facile Protein Engineering." - DSpace http://hdl.handle.net/1721.1/32535 (2006).
endogenous promoter for Fig1 (Factor Induced Gene 1)
Naringenin - chalcone synthase (CHS) coding region
Naringenin - chalcone synthase (CHS) coding region from ''Hypericum androsaemum''
Protein coding region for the NanoLuc(tm) Luziferase (designed by the Promega Corporation, free for research use), Codon optimized for yeast. The Nanoluc is smaller than other luziferase proteins and yields equal or stronger luminescence readings.
gBlock designed by Team Tuebingen, sequence by Promega
Partial DLD Promoter from Kluyveromyces lactis
Tais Sineiro Herig, Raissa Estrela Curado and Gleidson Silva Teixeira
Stable and soluble Histidine rich peptide. This part has a '''HindIII''' restriction site as extra.
PolyHb was desing taking into acount the following consideration: # avoided repeating the same residue in tandem to minimize local tRNA depleation # avoided Trp in tandem because of therir low solubility # we included Gly to avoid the formation of stable structures # included acidic and basic amino acids to increase solubility Sequence obtained by retro-translation. Codon and mRNA-secondary structure optimized for yeast.
OVA1 is an antigen expressed in tumor melanocytes cell line B16-OVA1. In the immune system, it is presented by the MHC-I to activate T-CD8 cells. It activated CD8+ in vivo against OVA1 in C57BL/6 mice, as tetramer CD8+ assays demonstrated.
OVAI was synthesized by the epitope SIINFEKL + surrounding residues to allow better processing by the immunoproteasome.
Yeast exportable Trp-rich peptide w/enhanced import (2)
Zif268-HIV bs + LexA bs + mCYC + Gli1 repressor (mCherryx2 tagged) on pRS303
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 12 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Gli1 binding sites + TEF constitutive yeast promoter
The Gli1 transcription factor binding sites on the TEF2 constitutive promoter. Transcription regulation should be constitutive plus regulated by Gli1-binding transcription factors.
Yeast optimized sfGFP N-terminus (1-214)
This is a E. coli promoter + Luciferase Composite part.
Note: The submitted version of this part has a mutation in the BioBrick suffix: The NdeI restriction site has been lost. However, the part can still be used, because the SpeI and PstI restriction sites are functional.
ECFP, yeast optimized for fusion proteins
Our BioBrick is cyan fluorescent protein which can be used to label proteins in yeast. Therefor it has specific characteristics of BioBrick part for fusion proteins (see design details).
We changed the format of the ECFP coding part of BBa_E2020 into Phillips/Silver standard.
Full Gli1 Repressor (tagged with mCherryx2)
Kozak+Sin3p RD+Gli1 DBD+mCherryx2+SV40NLS+tADH1
This is the same sequence as MFA1 promotor(BBa_K110008), but with suffix and prefix switched
Zif268-HIV bs + TEF2 + Untagged LexA activator on pRS306
S. pombe gene SPCC126.04c, coded for S. cerevisiae expression
SPCC126.04c is the pombe homolog of SGF73, a subunit of the SAGA ATP-independent chromatin remodelling complex in S. cerevisiae. Deletion of this subunit from S. cerevisiae leads to viable cells but poor expression of PAU genes under hypoxic conditions. Other phenotypes may also exist but are currently undescribed. The part BBa_Y00073 is the pombe SPCC126.04c ORF (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=68037958&itemID=1&view=gbwithparts) that has been codon optimized by DNA2.0 for cerevisiae expression. This may or may not be needed for optimal expression in cerevisiae, though the recoding does leave only 77% (794/1028) bp identities.
codon optimization for expression in new species
S. pombe gene recoded for S. cerevisiae expression
Sas2 histone acetyltransferase, Aar1 AB part
This is the coding for the Sas2 gene in yeast. It is a histone acetyltransferase (HAT).
Gli1 bs + TEF2 + Untagged LexA activator on pRS306
Microcystinase (Mlra) + mating factor alpha tag, for S. cerevisiae
pGAL1 + Null synthetic activator on pRS305
Note that this is a ligation that we made onto a pRS305 vector, which does not conform to Biobrick standard. As such, there is some remnant of the Biobrick ends but
4-coumarate - coenzym A ligase (4CL) + yeast consensus sequence
4-coumarate - coenzym A ligase (4CL) coding region from ''Arabidopsis thaliana'' preceeded by the yeast consensus sequence for improved expression.
enhanced version of EYFP, yeast-optimized YFP
yeast codon-optimized fast folding variant of YFP
Fusion protein of SpyCatcher and RPD3 in an expression cassette
Protein coding region for the cleavage site of the HCV NS3-4A protease (Part BBa_K1680011).
Composite part of BBa_K801031, Bba_K365005 and BBa_K801032
Essential Cys for chromophore attachement of phycocyanobilin (PCB)
Essential Arg for photoconvertible binding of PIF3
Essential Gly for photoconvertible binding of PIF3
Zif268-HIV bs + LexA bs + mCYC promoter
The Zif268-HIV and LexA transcription factor binding sites on the mCYC minimal promoter (in that order.) Transcription regulation comes from Zif268-HIV and LexA-binding transcription factors.
Yeast expression cassette for CaDXMT1 (BBa_K801072) controlled by the yeast TEF2 promoter (BBa_K801010) and the yeast ADH1 terminator (BBa_K801012)
Composite part of BBa_K801010, BBa_K801072 and BBa_K801012
Secretion tag from yeast α-factor mating pheromone (MFα1)
This part is the secretion signal peptide for the yeast α-mating factor. This signal peptide directs the secretion of the produced protein, and therefore allows for the exportation of it. This peptides are cleaved once the protein is in the lumen of the ER.
Prepro-a-factorHas a Cleavable Signal Sequence [Water et al 1987]
Yeast exportable His-rich peptide w/enhanced import (2)
promotor of the fet3 gene from S. cerevisiae, fet3 is repressed on high iron levels.
length of promoter was chosen to include binding sites and to obtain good primers
genomic upstream sequence of fet3 gene from the Saccharomyces Genome Database (http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=fet3)
Zif268-HIV binding sites + ADH1 yeast promoter
The Zif268-HIV transcription factor binding sites on the ADH1 constitutive promoter. Transcription regulation should be constitutive plus regulated by Zif268-HIV-binding transcription factors.
Zif268-HIV bs + TEF2 + Gli1 repressor (CFPx2 tagged) on pRS304*
Murine IFN gamma cytokine codon optimized to give the best yield in yeast S. cerevisiae. It promotes T cell activation, upregulates MHC-I and is pro-inflammatory.
This sequence was synthesised by IDT based on the genomic murine IFN gamma.
Protein coding sequence for a mutant (K145N) of the fluorescent protein Dronpa, codon optimized for Yeast. The mutation leads to photoswitchable monomerisation/multimerisation behaviour (as described by Zhou et al. 2012).
This promoter was isolated by taking 500bp upstream of the STE3 gene of S. cerevisiae. This promoter will be activated when alpha factor is expressed in an alpha haploid yeast cell.
Zif268-HIV bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS304*
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 9 AA linker as spacer (BBa_K1680001). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
Yeast Optimized sfGFP-C + ADH1 terminator + CaUra3 Cassette
Yeast exportable His-rich peptide w/enhanced import (1)
This composite device produces and exports a Histidine rich peptide, that is import enhanced (thanks to a trojan peptide). The high level structure of the device is: * Kozak consensus sequence for initiation of translation * Signal peptide that targets the product of the gene for secretion * Trojan peptide, to increase internalization in target cell * Payload: this is the exported amino acid rich domain of the protein
This is a targeting vector for homologous recombination into the ADE4 locus of S. cerevisiae. This vector can be used in cloning free cloning (ref. 2010 uOttawa iGEM wiki). Yeast strains with an ADE1 or ADE2 deletion or mutation have red colonies; by integrating the constructs using our protocol the ADE4 gene is knocked out and the colonies express a white phenotype.
pMET25 + Untagged LexA activator on pRS305
This is a composite part containing the minimal promoter mCYC preceded by multiple consecutive binding sites for the LexA binding domain.
Composite part was taken via PCR from a synthetic plasmid containing the sequence. The plasmid came from Lawrence Berkeley National Laboratory.
Aromatic prenyltransferase (APT) coding region
Aromatic prenyltransferase (APT) coding region from ''Humulus lupulus'', yeast codon optimized.
Synthesized by Geneart, yeast codon optimized http://www.ncbi.nlm.nih.gov/nuccore/AB543053
TDH3 Promoter + Yeast Kozak + FGFR-1/FRS2 Complex +TEF1 Terminator
 BioBrick Source & Functions: A. Promoter: BBa_124002; Yeast GPD (TDH3) Promoter Description: The Yeast GPD (TDH3) promoter was designed by John Szymanski of the 2008 Brown Group. This constitutive promoter is still in the planning phase and is 681 base pairs in length. B. Ribosome Binding Site: BBa_J63003; Yeast Kozak Sequence Description: The ribosome binding site was designed by Caroline Ajo-Franklin of the 2006 Silver Lab Group. The BioBrick presents a yeast kozak sequence (ribosome binding site) that is 18 base pairs in length. C. Coding Sequence: BBa_K976000; Fibroblast Growth Factor Receptor-1 Description: The Fibroblast Growth Factor Receptor-1 (FGFR-1) BioBrick has been designed by Amna Sohail and Meredith Gentry of the 2013 MCIT Indianapolis Group. This BioBrick presents a receptor that has a high affinity towards binding to the FGF2 ligand present upon the surface of potentially malignant melanocytes, and therefore may be utilized to detect presence of malignant melanoma tumor. The BioBrick itself is in the planning phase and is 2,466 base pairs in length. D. Coding Sequence: BBa_K976001; Fibroblast Growth Factor Receptor Substrate 2 Description: The Fibroblast Growth Factor Receptor Substrate 2 (FRS2) BioBrick has been designed by Amna Sohail of the 2013 MCIT Indianapolis Group. The Fibroblast Growth Factor Receptor Substrate 2, or FRS2 protein is a lipid based membrane-anchored docking protein that forms a fusion protein complex with the Fibroblast Growth Factor Receptor-1 (FGFR-1) to regulate downstream signal transduction. In melanocytes, this docking protein mediates signal transduction to pathways as the MAPK pathway that is involved in melanocyte proliferation, differentiation, and migration. FRS2 may be utilized to mediate downstream signaling to activate luciferase production after the FGF2 ligand binds to FGFR-1. This BioBrick is 1,524 base pairs in length. E. Terminator: BBa_K801011; TEF1 yeast terminator Description: The TEF1 Yeast Terminator was designed by Georg Sch??tzinger of the 2012 TU Munich Group. This BioBrick is derived as a PCR product of the genomic DNA sequence of Saccharomyces cerevisiae and is 507 base pairs in length.
Protein coding sequence for nuclear localization signal of SV40 large T antigen (amino acids: PKKKRKV).
Zif268-HIV binding sites + TEF constitutive yeast promoter
pTUM103 yeast shuttle vector with pADH1 promoter
This part corresponds to pTUM100 (BBa_K801000). The constitutive pADH1 promoter has been cloned in front of the MCS (RFC25 prefix and suffix).
Based on invitrogen's pYES2 plasmid. For more information see the part information of pTUM100 (BBa_K801000).
This is the coding sequence for the SSn8 gene in yeast. Ssn8 is involved in phosphorylation of the RNA polymerase C-terminal. It is involved in glucose repression as well as telomere maintenance. In our project Ssn8 is used as a "modifier" to enhance silencing.
RI7-odr10 (diacetyl) chimeric GPCR with promoter and terminator
BBa_K775003 encodes a chimeric diacetyl-sensing G-protein coupled receptor which can be functionally expressed in S. cerevisiae. This chimeric receptor is composed of the ligand-binding domain of the C. elegans 7-transmembrane receptor ODR-10, flanked by the N- and C- terminals of the rat OR RI7.
N-terminus: Olfactory receptor-like protein I7 (olr226), Rattus norvegicus (Rat) Ligand binding domain: C. elegans 7-transmembrane receptor ODR-10 U49449.1 C-terminus: Olfactory receptor-like protein I7 (olr226), Rattus norvegicus (Rat)
phenylalanine ammonia lyase (PAL) + yeast consensus sequence
phenylalanine ammonia lyase (PAL) coding region from ''Rhodosporidium toruloides'' preceeded by the yeast consensus sequence for improved expression.
Plasmid (template for PCR) provided by John A. Morgan, School of Chemical Engineering, Purdue University, West Lafayette, Indiana, USA http://www.ncbi.nlm.nih.gov/nuccore/AX366866 PMID: 1593299
Simon St. Pierre extracted from S. Cervisiae genome.
Gli1 bs + LexA bs + mCYC + LexA repressor (mCherryx2 tagged) on pRS306
S. pombe homolog of S.cerevisiae SGF29, recoded for expression in S.c.
Y00029 is S. pombe gene SPBC1921.07c [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein&cmd=search&term=NP_596000.2] recoded for expression in S. cerevisiae. The pombe gene is the homolog of the cerevisiae SGF29 [http://db.yeastgenome.org/cgi-bin/locus.pl?locus=sgf29], a subunit of the ATP-independent chromatin remodelling complex SAGA. Deletion of this gene in S. cerevisiae results in a viable strain. Requirements for SGF29 in SAGA function are largely unexplored. Unlike many of the SAGA subunits, the pombe and the cerevisiae gene encode proteins of abour the same size (259 aa in cerevisiae and 244 in pombe). The gene has been recoded for expression in S. cerevisiae by DNA 2.0, although it's not clear that this recoding will be necessary for expresssion. The recoded gene is 78% identical to the original pombe gene (Identities = 568/723).
insured no biobrick sites in ORF during optimization
DNA 2.0 optimization of pombe genomic sequence
shuttle vector pECPP11JS for recombination of genes of interest in yeast
the design considerations are coming soon
This part is made of the BioBricks XYZ by gibson assembly.
This is the coding sequence for the Sir2 gene with BD ends for Aar1 cloning. Sir2 is a HDAC that we use for silencing in our project.
AarI AD acceptor vector (pRS315, 8X LexAOps Fig1P, Adh1t)
Acceptor for AarI AD parts, with 8X LexA operators directly upstream of the medium strong Cyc1 promoter.
LexA Ops were cloned non-directionally into the PspOMI site. Inner site is broken, thus the cassette can be subcloned from this plasmid by PspOMI/SacI digestion (for example, to change markers).
Modification of the AarI AD acceptor vector (pRS315, Cyc1P, Adh1t).
3' UTR site of alcohol oxidase 1 gene (aox1)
3' UTR site of alcohol oxidase 1 gene (AOX1) from K. pastoris used as a recombination site.
The DNA sequence from plasmid pPIC9K of Invitrogen (http://products.invitrogen.com/ivgn/product/V17520?ICID==%3Dsearch-product) was used for primer design. But the genomic DNA of Pichia pastoris wildtype X33 was used for amplification via PCR.
This is a transcriptional repression domain. It can be fused to many DNA-binding domains to create synthetic transcriptional repressors. We have used it upstream of DNA binding domains.
From the yeast genome, via Caroline Ajo-Franklin.
Gli1 bs + ADH1+ LexA repressor (CFPx2 tagged) on pRS304*
AarI AD acceptor vector (pRS315, Cyc1P, Adh1t-8XLexA Ops)
This is a variant of the AarI AD acceptor vector (pRS315, Cyc1P, Adh1t), with the addition of 8X LexA operator sites directly downstream of the Adht1 terminator.
Variant of the AarI AD acceptor vector (pRS315, Cyc1P, Adh1t)
R.reniformis luciferase + ADH1 terminator + CaUra3
This is the promotor of yeast anb1 gene. Panb1 is a target of the rox1 repressor.
sequence and information from http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=SUC2
Cyc1p-mCherry-Adh1t-8xLexA Operators-Cyc1p-GFP-Adh1t
This is the GAL1 inducible promoter for use in Saccharomyces cerevisiae. It is repressed in the presence of glucose and induced in the presence of galactose. Raffinose is a neutral sugar source the neither induces nor represses the promoter. pGAL1 normally has an "all-or-nothing" response to galactose, but Christina Smolke has shown that knockout of the Gal2 gene gives pGAL1 a tunable, linear response to galactose.
From the Saccharomyces cerevisiae genome, via Caroline Ajo-Franklin.
Plasmid for Chromosomal Integration in Yeast at Ura3
This part is designed to allow simple chromosomal integration of BioBrick plasmids. It is based off of the bacterial pSB1AT3 backbone allowing for normal DNA manipulation to occur in bacteria. However the BioBrick cut sites are flanked by homologous regions to the Ura3 locus in S. cerevisiae. To transform S. cerevisiae the plasmid is cut with BbsI creating two non-sticky ends. The linear fragment then contains Ura3 homologous sequences at its ends that can be used to chromosomally integrate the BioBrick plasmid into the yeast genome. Transformants can then be selected for using Ura- medium.
This part was derived from plasmid pSB1AT3. Two successive rounds of PCR inserted a BbsI cut site and 40 bp homologous region in before the prefix and after the suffix. The following Primers were used: Forward 1 GCACAGAACAATAACCTGCTGGAAACGAAGATAAATCgaagacGATTACTTCGCGTTATGCAGGC Reverse 1 GCATCTTCTCAAATATGCTTCCCAGCCTGCTTATCcttctgAAATTCTGCCTCGTGATACGCC Forward 2 tactagtagcggccgctgcagTCTTAACCCAACTGCACAGAACAATAACCTGCTGGAAACG Reverse 2 ctctagaagcggccgcgaattcTTAGTATTGCTGGCCGCATCTTCTCAAATATGCTTCCCAGCC
mOrange, Yeast optimized for fusion proteins
Our BioBrick is red-orange fluorescent protein which can be used to label proteins in yeast. Therefor it has specific characteristics of a BioBrick part for fusion proteins (see design details). <br\n>
We changed the format of BBa_E2050 into Phillips/Silver standard.
Regulatory region spanning 680 bp upstream of the start codon of the GPD1 gene in yeast. Very strong constitutive promoter.
This is the coding sequence for the Sir2 gene with AB ends for Aar1 cloning. Sir2 is a HDAC that we use for silencing in our project.
pRS316 yeast shuttle vector with Biobrick MCS
Has an RFC10 compatible MCS, with ADH Terminator. No further RFC10 restrictions sites in the backbone.
This biobrick is the coding sequence of our SU9+REX in yeast cytoplasm. It is used to construct our final biobricks which express REX in the cytoplasm and then transport REX into mitochondrial matrix.
The signal peptid must be able to work efficiently, and can transport REX into mitochondrial matrix.
SU9 comes from yeast gemome. REX was modified to suitable for expression in yeast.
mig1 is a repressor targeting the promotor of suc2.
Sequence and information is based on the Saccharomyces Genome Database, see http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=mig1
This is a derivate of BBa_K105027. Due to a point mutation in the TATA box region the efficiency of this minimal promoter is only 28 % of the natural yeast CYC1 promoter.
This promoter was isolated by taking 500bp upstream of the MF(ALPHA)2 gene of S. cerevisiae. This promoter will be activated when alpha factor is expressed in an alpha haploid yeast cell.
This gene is on the crick strand of the entered sequence.
pTUM104 yeast shuttle vector with GAL1 promoter
This part is based on the plasmid pTUM100 (BBa_K801000). The galactose-inducible GAL1 yeast promoter has been cloned in front of the MCS (RFC25 prefix and suffix).
All forbidden restriction sites have been removed, making this plasmid RFC25-compatible.
mCherry, Yeast optimized for fusion proteins
Our BioBrick is red fluorescent protein which can be used to label proteins in yeast. Therefor it has specific characteristics of a BioBrick part for fusion proteins (see design details). <br\n>
We changed the format of BBa_E2060 into Phillips/Silver standard.
SV40NLS-PhyB-linker-Gal4DBD (part for GAL4 based light switchable promoter system)
Composite part of BBa_K801030, BBa_K801030, BBa_K365005, BBa_K801035
(+)-Limonene synthase 1 coding region of ''Citrus limon''. Improved version of BBa_I742111.
Coding sequence for Homo sapien Cripto-1 membrane protein.
Ryan Kawalerski, Tasnia Islam, Joshua Hombrebueno, Sarah Heacox, Samara Khan, Daniel Thach, Meghan Bialt deCelie.
Fusion protein of TALE3 and a SpyTag in an expression cassette
Gli1 binding sites + ADH1 constitutive yeast promoter
The Gli1 transcription factor binding sites on the ADH1 constitutive promoter. Transcription regulation should be constitutive plus regulated by YY1-binding transcription factors.
membrane progesterone receptor (Danio rerio)
Membrane progesterone receptor of Danio rerio. Upon ligand binding, the receptor represses expression of Fet3 in yeast.
restriction sites removed and codon usage optimized for yeast
rLucC + ADH1 terminator + CaUra3 cassette
R.reniformis luciferase + ADH1 terminator + Kanamycin resistance
The kozak sequence acts as a eukaryotic RBS. It is cloned directly between a promoter and coding region.
Strong Expression level Constitutive promoter in yeast
Endogenous promoter for Alcohol Dehydrogenase
Alpha amylase with mating factor alpha secretion
This is the alpha amylase from Bacillus amyloliquefaciens, flanked by Kozak sequence (Part BBa_K165002), mating factor alpha secretion sequence (Part BBa_K792002), and the ADH1 Terminator (Part BBa_K392003). This part is contained by the minimal cyc promoter (Part BBa_K105027).
This is derived from the published sequence for Bacillus amyloliquefaciens.
Native Mating Factor Alpha Secretion Sequence
Fusion protein of the TALE constant domain and a SpyTag in an expression cassette
Kozak+Sin3p RD+LexA DBD+CFPx2+SV40NLS+tADH1
LacI promoter with RBS, followed by OmpA outer membrane protein and linker for Escherichia coli cell surface display.
Composite part BBa_K392001 (LacI promoter + RBS) and registry part BBa_K103006 (OmpA w/ linker).
pYES2 - galactose-inducible expression vector for yeast
membrane progesterone receptor (Xenopus laevis)
Membrane progesterone receptor of Xenopus laevis. Upon ligand binding, the receptor represses expression of Fet3 in yeast.
Fusion protein of TALE1 and a SpyTag in an expression cassette
mCYC promoter plus Zif268-HIV binding sites
This is a composite part containing the minimal promoter mCYC preceded by multiple consecutive binding sites for the Zif268-HIV binding domain. It contains the sequences of parts BBa_K165011 and BBa_K165016 but also contains additional DNA.
Constitutive yeast promoter of medium strength compared to TEF1 promoter.
The pre-region of lipase 2 from Yarrowia lipolytica corresponds to the signal sequence; the dipeptides XA and XP are substrates for diamino-peptidase; the dibasic KR cleavage site is substrate for Xpr6p endoproteinase.
pTUM102 yeast shuttle vector with pTEF2 promoter
AarI AD acceptor vector (pRS315, Cyc1P, Adh1t)
Acceptor vector for AarI based cloning. Accepts any combination of parts beginning with an A site and ending with a D site. Expression of construct is driven by a Cyc1 promoter, with Adh1 terminator.
The pRS315 vector has been modified with an AarI cloning cassette inserted between the PspOMI and SacI sites. Please note, the actual orientation of this vector is reversed.
A modified pRS315 plasmid--CEN ARS yeast expression vector (LEU2 marker).
This composite part consists of the Aga2 yeast surface display CDS linked to a flexible (Glycine3Ser)4 linker. It can be used by linking it upstream of the protein you wish to be displayed on the surface of the yeast cell. Note: Aga1 must be constitutively expressed by the yeast strain for Aga2 to anchor to the cellular surface.
This composite part was put together following RFC23, thus the scar between parts consists of the sequence ACTAGA
This part comes from the Aga2 [K416000] and Linker [K416001] parts we constructed this year.
Chalcone O-methyltransferase (OMT1) + yeast consensus sequence
Chalcone O-methyltransferase (OMT1) coding region from ''Humulus lupulus'' preceeded by yeast consensus sequence for improved expression.
low expression, constitutive promoter in yeast
This is a low-level constitutive yeast promoter.
This minimal promoter can be used as a base for designing promoters by adding binding sites upstream.
PKI nuclear export sequence; yeast codon optimized
yeast codon optimized PKI nuclear export sequence
xanthosine methyltransferase CaXMT1-strep
xanthosine methyltransferase CaXMT1-strep from ''Coffea arabica'' followed by a strep-tag for purification and/or detection via western blot.
http://www.ncbi.nlm.nih.gov/nuccore/AB048793 synthesized by IDT, yeast codon optimized
Plasmid for Chromosomal Integration in Yeast at His3
This part is designed to allow simple chromosomal integration of BioBrick plasmids. It is based off of the bacterial pSB1AT3 backbone allowing for normal DNA manipulation to occur in bacteria. However the BioBrick cut sites are flanked by homologous regions to the His3 locus in S. cerevisiae. To transform S. cerevisiae the plasmid is cut with BbsI creating two non-sticky ends. The linear fragment then contains His3 homologous sequences at its ends that can be used to chromosomally integrate the BioBrick plasmid into the yeast genome. Transformants can then be selected for using His- medium.
KlADH4 yeast promoter, ethanol inducible
This part is the ethanol inducible promoter controlling the KlADH4-gene of ''K. lactis''.
Extracted from genomic DNA of ''K. lactis'' using PCR.
possible transcription initiation region (Inr)
This is a mid-high level constitutive promoter from the yeast gene TEF2.
This is the same sequence as STE2 promotor(BBa_K110008), but with suffix and prefix switched
Zif268-HIV bs + LexA bs + mCYC + Zif268-HIV repressor (mCherryx2 tagged) on pRS306
Pichia pastoris initially gained attention as an expression system after recognition of its methylotrophic capabilities and the high levels of alcohol oxidase(AOX) present when induced by methanol. By isolating the promoter (pAOX1 )responsible for AOX production, this system can be utilized to obtain high yields of heterologous protein. The promoter provides not only high expression but very tight regulation as well. Vectors providing protein expression under the control of pAOX1 are the primary system used for P. pastoris
This part was obtained by designing primers and isolating it from the organisms DNA. There were no unwanted restriction enzyme sites to eliminate
This promoter was isolated by taking 500bp upstream of the MF(ALPHA)1 gene of S. cerevisiae. This promoter will be activated when alpha factor is expressed in an alpha haploid yeast cell.
Yeast Optimized sfGFP-N + ADH1 terminator + Kanamycin resistance
Psuc2 promotor from yeast. Repressed by mig1
mRFP derivative. Ex548nm/Em562. yeast codon optimized.
Contains N- and C-terminal linker sequences (with homology to biobrick parts BBa_E2060 (mCherry), BBa_E2020 (Cerulean CFP), and BBa_E2030 (Venus YFP) to facilitate color swapping in yeast. Adds N-"MATSG" and "GSGTA"-C. to published amino acid sequence. Double TAATAA stop codon. Missing EcoRI, HindIII, NotI, NdeI, XhoI, RsrII, BamHI, NcoI, BglI, SpeI, XbaI, and PstI. except for 5' and 3' ends no significant sequence identity runs to mCherry (BBa_E2060).