Contains both an early MfeI site and a late NsiI site. Cutting with Mfe and Spe and ligation to a BB part cut EX will form two mixed sites, neither of which can be recut. Cutting with Xba and Nsi and ligation to a BB part cut SP will again form two mixed sites, neither of which can be recut.
Should be grown in pSB1A2, as Nsi cuts both pSB2K3 and pSB1AK3 and Mfe cuts pSB4A3. 70 bp from Xba to Nsi or Mfe to Spe, the minimum for gel extraction. The middle is largely noise, to raise the size of the part.
Obtained by PCR from the genome of Pseudomonas putida KT2442.
Codifies the LapG protein from Pseudomonas putida KT2442, a periplasmic protease that participates in the biofilm dispersion process by cleaving adhesion proteins such as LapA.
We have inlcuded the sequence corresponding to RBS BBa_B0034 with the PCR primers used for its amplification, as well as the scar it should have been produced by the clonning of both parts
OriT-R (Origin of transfer for the R-plasmid nic region)
OriTR, the R plasmid nic region, is where the relaxosome nicks the plasmid and conjugative transfer by R plasmid machinery begins.
Shortened RNAse E site (Nilsson et al. 1996) plus shortened insertion site (I13452)
Emahi et al. 2014 (http://www.rsc.org/suppdata/ra/c4/c4ra11052h/c4ra11052h1.pdf)
This is a functional DNA sequence that encodes the pyrilloquinone quinone binding aptamer. It can be used in both binding and sensing applications.
This was one of the best two DNA aptamer candidates from SELEX (also see BBa_K2027050).
RNAse E site (Smolke et al., 2002) followed by insertion site (shortened I13452)
KpnI restriction enzyme site with extra flanking nucleotides
KpnI restriction site with 3bp flanking each side
This is a part consisting of only a single nucleotide ... G. It can act as a spacer between restriction sites and parts.
ApaI restriction site with extra flanking nucleotides
Oligo 3 of DNA Tetrahedral Nanostructure
This will be used to build our desired nanostructure that target micro RNA biomarkers. This plasmid encodes the second ssDNA that will make the tetrahedral nanostructure, together with oligo 1, 2, 4 and 5.
Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7.
We use this sequence to insert it into staples as a level 2 pixel.
OligoAnalyzer 3.0 http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
BioScaffold Linker - Removes Stop Codons & scars & replaces with a Gly-Ser linker
This part is completely designed de novo. This sequence does not occur naturally.
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein** ====N.B.==== CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion. The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. Updates will soon follow on this. This version accounts for cleaving DNA 12/10bp (top/bottom) on the 5' of the recognition sequence and 10/12bp (top/bottom) on the 3' site.
CspCI enzyme has different variations in the distance of restricting DNA from the recognition site.This biobrick is almost identical to part BBa_K259004 but modified to account for this discrepancy.
This part is the cDNA of a mutated calcium ion channel protein.
This part encodes the gene of Calcium release-activated calcium channel protein 1 (Orai1), which is a part of calcium selective ion channel in humans. Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE).Store-operated calcium (Ca2+) entry (SOCE) is activated in response to cellular stimuli that deplete the sarco/endoplasmic reticulum (ER) lumen of Ca2+, a prerequisite event that opens exquisitely Ca2+ selective plasma membrane (PM) channels, augmenting cytosolic Ca2+ levels and refilling ER stores. The principal molecular components of SOCE in many cell types include the ER-inserted stromal interaction molecule-1 (STIM1)3,4 and the Orai1 PM channel subunits5,6,7,8, which physically interact at ER???PM junctions in a functional Ca2+ release-activated Ca2+ (CRAC) channel complex. SOCE through CRAC channels is integral to myriad signalling pathways in electrically excitable and non-excitable cells, the most prominent being immune cells in which inheritable mutations in STIM1 and Orai1 have been linked to devastating immunodeficiency diseases. Therefore, we use this part to help with eliminating Ca2+ in hard water.
Usually, the protein Orai1 works only when the STIM1 channel is activated. However, we select the mutated protein that did not require the presistence of STIM1, which changed the 102th amino acid valine to alanine.
This part comes from the cDNA of mutated Orai1 protein (V102A).
OriT-R (Origin of Transfer for the R751-plasmid nic region)
Enterobacter aerogenes plasmid R751 NCBI:NC_001735
OriT-R751 (Origin of Transfer for the R751-type conjugative plasmid) is the nic region, where the relaxosome nicks the plasmid and conjugative transfer by R751 plasmid machinery begins via rolling circle replication.
T50 is the thrombin-binding component of the A50 adaptamer which can physically link together the proteins thrombin and streptavidin.
Linking region had to avoid base-pairing with aptamer sequences.
A BioScaffold Part (Uses AarI) for Library Insert Preparation (see Part Design page)
When placed in a BioBrick assembly and cut with the restriction enzyme AarI, this part will direct the restriction enzyme to cut within the BioBrick scar still in testing, uses AarI (2) XmaI (1) See part design page or BBFRFC15, for details about use
This part was designed ''de novo''. The sequence does not occur naturally.
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions==== *Create an Assembly Standard 10 fusion giving ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part *Use the BioScaffold Specific Enzymes **Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. **Ligate and you will get the intermediate product; Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein **Restrict with BseRI to remove downstream scar. **Ligate to get the final product; Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein
Part was designed taking into account RFC10 assembly standard and as an attempt to be upwards compatible with RFC10.
Tyr Residue involved in Restriction/Ligation
The sequence codes the scar, that is introduced between two parts, ligated together after being cut with BspEI/NgoMIV.
This sequence is introduced as scar as consequence of ligation of two parts, that were previously cut with BspEI/NgoMIV.
This is a part consisting of only a single nucleotide ... A. It can act as a spacer between restriction sites and parts.
A15 is a single-piece adaptamer which which can physically link together the proteins thrombin and streptavidin.
Linking region had to avoid disruption of aptamer secondary structure.
T20 is the thrombin-binding component of the A35 adaptamer which can physically link together the proteins thrombin and streptavidin. A20 contains a 15 bp double-stranded linking region between thrombin and streptavidin aptamer sequences.
Coding Sequence of IscA1 pigeon homologue (codon-optimized for humans)
Michael Becich, Thomas Lau, Sreyas Misra
The gene Iron-Sulfur cluster assembly 1 (IscA1) is a mitochondrial protein involved in the biogenesis and assembly of iron-sulfur clusters, which play a role in electron-transfer reactions and is theorized to be associated with certain organisms' magnetic perception. The homing pigeon is one such organism with this ability, and here we have codon-optimized the Columba Livia homologue for homo sapiens. The sequence can be inserted into plasmids for expression of the magnetoreceptor IscA gene, which hopefully will create magnetogenetic biosensors in the form of iron-sulfur cluster, for downstream output and applications.
While the plasmid design for this insert can be complicated, the coding sequence itself is quite simple. We only needed to remove the start codon in order to co-express a fluorescent protein, and modify two BbsII restriction enzyme sites by altering the third base pair of codons (wobble effect) at bp locations 75 (C->G) and 309 (A->G).
The predicted IscA amino acid sequence was obtained from NCBI (http://www.ncbi.nlm.nih.gov/protein/XP_005508102.1), and codon-optimized for humans with IDT's tools.
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.
SalI restriction enzyme site with extra flanking nucleotides
Encryption key sequence in integration plasmid (BBa_K823023)
Key sequence: Created by our CryptoGErM software. http://2016.igem.org/Team:Groningen/Encoding BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The key sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted key sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our key sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation.
Nucleotide sequence of a cDNA clone of saxiphilin from the liver of Rana Catesbeiana
It comes from the cDNA corresponding to the RNA of the bullfrog (Rana Catesbeiana) liver.
This is the nucleotide sequence of a cDNA clone of saxiphilin from the liver of the American Bullfrog, Rana Catesbeiana. Saxiphilin is a protein that binds a neurotoxin called saxitoxin. It is a non-membrane bound binding site and is considered to be a member of the transferrin family of plasma proteins.
Since it is a copy of a cDNA derived from RNA that is already spliced, the sequence only reflects the exon parts, not the introns.
trbK (entry exclusion) of IncP beta R751 plasmid
This sequence can be found at http://www.ncbi.nlm.nih.gov/protein/10955220
This part encodes for trbK, a small 75 amino acid cytoplasmic membrane lipoprotein which handles the entry exclusion in the R751 plasmid. Expression of this part in a cell without R751 will significantly reduce the transfer frequency from R751 donors. This is designed to prevent redundant transfers from occurring. Note that the presence of entry exclusion has been linked to the prevention of lethal zygosis. If the entry exclusion (Eex) index is defined as the transfer frequency for receiver cells not containing the trbK protein divided by the transfer frequency for receiver cells containing the trbK protein than for R751 Eex index = 44,000 (See Table 5 of Haase, J., Kalkum, M. & Lanka, E. TrbK, a small cytoplasmic membrane lipoprotein, functions in entry exclusion of the IncP alpha plasmid RP4. J. Bacteriol. 178, 6720???6729 (1996).[http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=178567])
This is the sequence of the SpeI/XbaI scar for RBS-CDS junctions in BioBricks standard assembly.
This is a shorter scar to ensure proper spacing between the RBS and CDS.
An E. coli strain that carries the cloned Acc65I gene from Acinetobacter calcoaceticus 65
OriT-RP4 (Origin of transfer for the RP4 plasmid)
Relaxation region of self-transmissible plasmid RP4. When this part is added to a plasmid, it is transferred by RP4 through conjugation.
This part is similar to J01003, which has no experience and is much longer. In the process of designing and testing this part, it will be compared to J01003.
T35 is the thrombin-binding component of the A35 adaptamer which can physically link together the proteins thrombin and streptavidin.
Coding Sequence of IscA2 pigeon homologue (codon-optimized for humans)
The gene Iron-Sulfur cluster assembly 2 (IscA2) is a mitochondrial protein involved in the biogenesis and assembly of iron-sulfur clusters, which play a role in electron-transfer reactions and is theorized to be associated with certain organisms' magnetic perception. The homing pigeon is one such organism with this ability, and here we have codon-optimized the Columba Livia homologue for homo sapiens. The sequence can be inserted into plasmids for expression of the magnetoreceptor IscA gene, which hopefully will create magnetogenetic biosensors in the form of iron-sulfur cluster, for downstream output and applications.
While the plasmid design for this insert can be complicated, the coding sequence itself is quite simple. We only needed to add the start codon since this second isoform did not have methionine built in.
The predicted IscA amino acid sequence was obtained from NCBI (http://www.ncbi.nlm.nih.gov/protein/XP_005502393.1), and codon-optimized for humans with IDT's tools.
This will be use to build our desired nanostructure. This ssDNA contains also split G-quadruplex sequences that allow the oligo itself to function as an DNAzyme under specific conditions.
Plasmid Construction: Elbaz, J., Yin, P., & Voigt, C. A. (2016). Genetic encoding of DNA nanostructures and their self-assembly in living bacteria. Nature communications, 7. Oligo Construction: Nakatsuka, K., Shigeto, H., Kuroda, A., & Funabashi, H. (2015). A split G-quadruplex-based DNA nano-tweezers structure as a signal-transducing molecule for the homogeneous detection of specific nucleic acids. Biosensors and Bioelectronics, 74, 222-226.
Left end of a linear transpososome sequence containing the 19 bp mosaic end in the forward orientation. The sequence also contains a PvuII cut site which releases the transpososome when cut with PvuII. Constructed compound parts intended for transposition must not contain other PvuII end, or be PCR amplified with blunt end producing enzymes (Pfu, Phusion) at the PvuII cut site between CAG and CTG. This part, and the companion part M1001 provide Biobrick tools to construct Tn5 transpososomes: linear DNA sequences which can be cut in vitro from a plasmid backbone, or created by PCR, combined in vitro with purified Tn5 trasposase protein, electroportated into cells, and finally randomly inserting into the bacterial chromosome. The parts assembled between the two mosaic ends can be delivered, including antibiotic resistance markers or other functional chromosomal insertions.
;biobrick left end, TA cloning overhang GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG ;biobrick right end, TA cloning overhang GTT TCT TCC TGC AGC GGC CGC TAC TAG TA ;mosaic end with cag upstream to insert PvuII cut site cagCTGTCTCTTATACACATCT ;mosaic end reverse complement with ctg upstream to insert PvuII cut site AGATGTGTATAAGAGACAGctg ;mosaic left end in biobrick form construction primer F GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG cagCTGTCTCTTATACACATCT, MEL-F ;mosaic left end in biobrick form construction primer R GTT TCT TCC TGC AGC GGC CGC TAC TAG TA AGATGTGTATAAGAGACAGctg, MEL-R ;mosaic right end in biobrick form construction primer F GTT TCT TCG AAT TCG CGG CCG CTT CTA GAG AGATGTGTATAAGAGACAGctg, MER-F ;mosaic right end in biobrick form construction primer R GTT TCT TCC TGC AGC GGC CGC TAC TAG TA cagCTGTCTCTTATACACATCT, MER-R
A BioScaffold Part (Uses PpiI), for offset see Part Design page
NheI/SpeI mixed site. Used to help enter parts into the registry.
Encrypted message sequence in integration plasmid (BBa_K823023)
Message sequence: Created by our CryptoGErM software. http://2016.igem.org/Team:Groningen/Encoding BBa_K823023: Created by iGEM Munich 2012. http://parts.igem.org/Part:BBa_K823023
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The message sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted message sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our message sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation.
The iGEM team Groningen 2016 worked on bioencryption in order to safely store data in DNA. In our project we were mainly working with two sequences of DNA which were the encrypted message sequence and the encryption key sequence. The message sequence was created by our software. It is therefore artificial DNA. We ordered the encrypted message sequence as gBlock from IDT (Integrated DNA technologies). This part was created in the interest of having a genomic integration plasmid with our message sequence. We used an available BioBrick BBa_K823023 from igem Munich 2012. It is an integration plasmid for Bacillus subtilis, which can be used for cloning in E. coli as well. An RFP is inserted in BBa_K823023 for more efficient screening after transformation. For more information see plasmid construction under our lab journal: http://2016.igem.org/Team:Groningen/Experiments
This is a '''BamHI'''restriction site. This part might not be useful as a physical biobrick. It has been created to follow modularization in parts X
AvrII restriction enzyme site (XbaI, SpeI compatible overhang)
AvrII recognition site. Digestion with AvrII generates a compatible cohesive end to XbaI and SpeI.
The DNA was amplified from E. coli BL21 genomic DNA using primers based on published sequence (Genbank accession J01636, gi:146575). The annotation shown here is based on that associated with this Genbank entry. The sequence shown here is derived by sequencing the construct.
This part (submitted in pSB1A2) consists of the lac promoter and lacZ' gene, encoding the N-terminal 76 amino acid residues of LacZ, sufficient to complement the lacZ-delta-M15 mutation for blue-white selection on Xgal plates. A SacI site has been introduced at the 3' end, overlapping the XbaI site of the Biobrick prefix. This is designed to be used as a cloning vector for making new biobricks. PCR primers can be designed with a SacI site in one primer and an SpeI site in the other. This removes the necessity for an excessively long non-complementary tail on one primer bearing either the full biobrick prefix or suffix. The PCR product can then be digested with SacI and SpeI for insertion into this plasmid, replacing the Plac-lacZ' cassette. Recombinant plasmids will then be white on IPTG/Xgal plates, whereas any that still contain the original insert will be blue. We have used this strategy to prepare several biobricks, including BBa_J33204, which contains the xylE gene encoding catechol-2,3-dioxygenase. (For making biobricks that contain lacZ', BBa_J33204 can be used in the same way; in this case, clones with plasmids that still contain xylE will turn yellow on addition of a drop of 10 mM catechol.)
Note that the SacI site overlaps the SpeI site. The Biobrick prefix ends ...TCTAGAG. When this is added to the CTC at the start of the sequence shown here, the SacI site, GAGCTC, is generated.
OriT-F (Origin of Transfer for the F-plasmid nic region)
Origin-of-transfer nic region excised from Plasmid F genomic DNA.(NCBI:AP001918)
OriT-F (Origin of Transfer for the F-type conjugative plasmid) is the nic region, where the relaxosome nicks the plasmid and conjugative transfer by F plasmid machinery begins via rolling circle replication.
This OriT-F is the same as the host cell's native Origin of Transfer, Please compare the sequence with J01002.
This is the 6 bp mixed site that results when ligating the compatible ends (CTAG 5' overhang) that result from AvrII cleavage (CCTAGA) of the upstream part and XbaI (TCTAGA) cleavage of the downstream part.
A BioScaffold Part (Uses PsrI), Protein Head Remover see Part Design page
This part is just for upload test. It is meaningless
XbaI restriction site with extra flanking nucleotides
MfeI restriction enzyme site (EcoRI compatible overhang)
MfeI recognition site. Digestion with MfeI yields a compatible cohesive end to EcoRI.
PvuI restriction enzyme site with extra flanking nucleotides
PvuI restriction site with 3 flanking base pairs
Linker for BioScaffold Parts, example of Gamma BioScaffold part
Will use as a linker when fusing two BioScaffold parts, can be created as an oligo
The pUC18-SfiI cloning vector was kindly provided by the Microbiology laboratory of Centro Andaluz de Biolog??a del Desarrollo (CABD) in Sevilla. The miniTn7BB-Gm synthetic transposon was designed by the iGEM 2011 team UPO-Sevilla based on the set of miniTn7 derivatives developed by Choi et al. (2005) and commercially synthesized by Mr. Gene (Regensburg, Germany). K.-H. Choi, J. B. Gaynor, K. G. White, C. Lopez, C. M. Bosio, R. R. Karkhoff-Schweizer & H. P. Schweizer (2005). A Tn7-based broad-range bacterial cloning and expression system. Nature Methods, vol.2 NO.6, June 2005, 443.
The miniTn7BB-Gm synthetic minitransposon was digested at the flanking SfiI sites and cloned into SfiI-digested pUC18-Sfi. This results in the elimination of the complete pUC18-SfiI multi-cloning site, except for the duplicated SfiI sites that remain on both sides of the transposon, thus facilitating its transfer to other vectors. This plasmid can be used to keep the mini-Tn7-Gm in E. coli and other enterics, and may be used for transposition into the genomes of non-enteric bacteria, in which it is non-replicative. The structure of the mini-Tn7BB-Gm transposon is shown in the figure below. Tn7R and Tn7L are the right and left ends of Tn7 transposon, respectively, required for recognition of the transposase machinery and transposition. FRT is the Flp recombinase target site for excision of the resistance once the transposon is inserted in the genome of a living organism. The gentamycin resistance cassette (Gm) is flanked by restriction sites in order to facilitate the generation of variants with different antibiotic resistance markers. The BioBrick cloning site (BCS) includes the prefix and suffix restrictions sites used in assembly standard 10, and it is the place where BioBricks are inserted for genome integration. The complete construction is flanked by SfiI restriction sites to facilitate cloning.
In order to make this vector compatible with assembly standard 10, several restriction sites were removed from the synthetic sequence.
This is a part consisting of only a single nucleotide ... C. It can act as a spacer between restriction sites and parts.
This is the scar sequence of the BBb1 assembly scheme. For more information, refer to [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page].
SapI restriction enzyme site (offset cutter)
SapI is an offsite cutter restriction site. The recognition sequence for this enzyme is the 8 bp sequence GCTCTTCN (on the 5' strand). Cleavage at this site results in a 3 bp, 3' overhang (right side, bottom strand) directly downstream of this sequence. This specific part has a T in the 8th position.
This part was designed for use with Heather Keller's alternative assembly method for blunt cloning RBS sequences upstream of a coding sequence.
NheI restriction enzyme site (XbaI, SpeI compatible overhang)
NheI recognition site. Digestion with NheI generates a compatible cohesive end to XbaI and SpeI.
It was obtained by PCR from a plasmid containing this sequence. The PCR primers added the prefix and suffix needed.
nahR (Pseudomonas putida, see BBa_K1031610) is expressed under the constitutive Pr promoter. It encodes a regulatory protein that activates the Psal promoter (see BBa_J61051) in the presence of salicylate.
We had included the RBS BBa_B0034 in the PCR primers, as well as the scar it should have appeared in the cloning of the DNA piece.
AclI restriction enzyme site with extra nucleotides
AclI restriction site with 3 flanking bp
A BioScaffold Part (Uses AarI) for Library Vector Preparation (see Part Design page)
OriT-S (Origin of Transfer for the pSC101-plasmid nic region)
OriT-S (Origin of Transfer for the pSC101-type conjugative plasmid) is the nic region, where the relaxosome nicks the plasmid and conjugative transfer by pSC101 plasmid machinery begins via rolling circle replication.
S35 is the streptavidin-binding component of the A35 adaptamer which can physically link together the proteins thrombin and streptavidin. A35 contains a 30 bp double-stranded linking region between thrombin and streptavidin aptamer sequences.
BmtI restriction enzyme site with extra flanking nucleotides
BmtI restriction site with three base pairs before site
A BioScaffold Part (Uses AarI, MabI) for Library Vector Preparation (see Part Design page)
When placed in a BioBrick assembly and cut with the restriction enzyme AarI, this part will direct the restriction enzyme to cut within the BioBrick scar still in testing, uses AarI (2), MabI(2), XmaI (1) * XmaI does not work in vector for BioScaffold part manipulations See part design page or BBFRFC15, for details about use
See BBa_J70034, this is an analogous part except it uses MabI
Low to medium copy Lambda Red recombineering compatible plasmid
The P22 late terminator come from the genome of the Salmonella phage P22 (GenBank: AF217253.1, 19662..19702), and the kanamycin resistance marker flanked by FRT sites comes from the pKD4 plasmid (Datsenko and Wanner, 2000).
BBa_K592202 is a BioBrick friendly vector with low to medium copy p15A replication origin (BBa_I50032) and a kanamycin antibiotic resistance marker, usable for Lambda Red recombineering in E coli. The E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22, and the kanamycin resistance marker is flanked by FRT sites so that the resistance marker can be removed after chromosomal integration using the FLP recombinase.
To make the plasmid usable for Lambda Red recombineering, all sequences with homologies to the E coli chromosome had to be removed. To accomplish this, the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. To be able to remove the antibiotic resistance marker after chromosomal integration, the kanamycin marker from the pKD4 plasmid (Datsenko and Wanner, 2000) was used. The resistance marker can be removed after chromosomal integration using the FLP recombinase, leaving only the plasmid insert and a scar behind.
MluI restriction enzyme site with flanking nucleotides
Contains an MfeI restriction site. Cut this part with MfeI+Spe and the other part with Eco+Xba. Ligation will form two mixed sites, neither of which can be recut. This effectively fixes the left side of the Biobrick part
The oriT sequence was taken from the self-transmissible plasmid RP4. LacZ is available as a part in the registry.
This part was constructed to compare the oriTr, which is currently available in the registry to oriT which was recently designed to perform the same function. The oriTr was added to LacZ part so that conjugation could be monitored by blue/white selection.
The oriT was synthesized and J33207 was extracted from the registry.
Oligo 2 of DNA Tetrahedral Nanostructure
This will be used to build our desired nanostructure. This plasmid encodes the second ssDNA that will make the tetrahedral nanostructure, together with oligo 1, 3, 4 and 5.
A BioScaffold Part (Uses PpiI), Protein Tail Remover see Part Design Page
When placed in a BioBrick assembly and cut with the restriction enzyme PpiI, this part will direct the restriction enzymes to cut outside the BioBrick scar still in testing, uses restriction enzyme sites PpiI (2) PacI (2) MabI (2), will place in pSB1AK3 vector
Can be used to removed protein tails for protein fusions, see BBF RFC 15 XXXXX^ XXX TACTAGAG BioScaffold Part BBa_J70030 TACTAGAT XXXXX^ ^XXXXX XXX ATGATCAC ATGATCTA^XXXXX XXXXX^ TAA TACTAGAG BioScaffold Part BBa_J70030 TACTAGAT XXXXX^ ^XXXXX ATT ATGATCAC ATGATCTA^XXXXX
NsiI restriction enzyme site (PstI compatible overhang)
NsiI recognition site. Digestion with NsiI generates a compatible cohesive end to PstI.
This DNA will be used as a part of DNA-origami device. The mechanical movement will be performed by B???Z DNA transition in the presence/absence of Co(NH3)6Cl3.
Mao C, Sun W, Shen Z, Seeman NC. A nanomechanical device based on the B-Z transition of DNA. Nature. 1999 Jan 14;397(6715):144-6.
This part is the standard BioBricks prefix.
S20 is the streptavidin-binding component of the A20 adaptamer which can physically link together the proteins thrombin and streptavidin. A20 contains a 15 bp double-stranded linking region between thrombin and streptavidin aptamer sequences.
Basic parts were cloned into the miniTn7BB-Gm Backbone
nahR (Pseudomonas putida, see BBa_K1031610) is expressed under the constitutive Pr promoter. It encodes a regulatory protein that activates the Psal promoter (see BBa_J61051) in the presence of salicylate. lapG (see BBa_K1973000) is expressed from the Psal promoter in the presence of this molecule. The miniTn7 tool allows the stable integration of this module in the chromosome of a wide range of bacteria (see http://parts.igem.org/Genome_Integration).
Designs are described in basic parts pages
Muscarella,D.E., Ellison,E.L., Ruoff,B.M. and Vogt,V.M. (1990) Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum. Mol. Cell. Biol., 10, 3386???3396.
Restriction site for the homing endonuclease I-PpoI.
Randomly generated and optimized for several parameters (see Design notes).
Generic spacer for ensuring a 70 bp distance between the end of the suffix of the BioBrick part containing the double terminator and the prefix of the BioBrick part containing the promoter of the new gene. Please, use the AlignX function of Vector NT to check for homology with the components in your plasmid before using this spacer.
The size of the spacer was choosed to meet the minimum length of a sequence that can be queried using the BLAST search engine. However, subsequences of it can be used to design shorter spacers. The sequence was selected from many more sequences randomly generated using the Random DNA Generator engine; the GC% parameter used as input was 50%. The sequences were selected based on the following constraints listed in their order of importance: the absence of any putative promoter regions, a low degree of homology with the Elowitz plasmid (whose components are widely used in our designs), no homology with other E.coli sequences as shown by BLASTN search results and the presence of a number of TAA stop codons. The second constraint was the most stringent leading to the elimination of most sequences. DE made the following changes to the original sequence in order to add stop codons in the -3 frame and more in the +2 frame (note, not all of these stop codons are UAA. Thus, if used in an organism that inserts an amino acid @ UGA or UAG the obvious will occur): T->A @ 85 T->A @ 42 C->T @ 79 A->T @ 64 A->T @ 31 T->A @ 34 C->A @ 37 Also, note that the above changes further reduce (the already very weak) homology to current NCBI-stored sequences. In the process of selecting the best sequence it appeared that a good alternative sequence for a spacer would be: AGGTTCTGATATGTAACTGTGCCCAATGTCGTTAGTGACGCATACCTCTTAAGAGGCCACTGTCCTAACA. The sequence contains no putative promoters and shows moderate homology with the 5' end of the Ampicillin resistance gene. However a strong promoter sequence starts 12 bp downstream of this sequence, and therefore the sequence presented above was preferred. The sequence is compatible (does not show significant homology) with the components in the Elowitz repressilator plasmid.
Vinay S. Mahajan, Brian Chow, Peter Carr
This part was built from oriTr and LacZ.
This part will be tested by blue/white screening.
All Oligos for simplified Tetrahedral Nanostructure
This will be used to build and express our desired functional nanostructure in E. coli that target micro RNA biomarkers, but a simplified 2-strand nanostructure. This plasmid encodes the two ssDNA that will make the nanostructure, i.e. Oligos 1 and 5, from parts BBa_K2054001 and BBa_K2054005 respectively.
software generated kanamycin aptamer (candidate 2)
Aptamer binding kanamycin created by the MAWS Software from iGEM Team Heidelberg
MAWS software from iGEM Team Heidelberg (created with the pre-alpha version)
For further details on the in silico aptamer generation see:
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.
This part is completely designed ''de novo''. This sequence does not occur naturally.
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein**
This part was designed taking into consideration the requirements by Assembly Standard 10 (RFC10).
I-SceI homing endonuclease site Restriction site for the homing endonuclease I-SceI. Sequence will be cut as follows: 5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3' 3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5'
Design was very simple. The recognition site was copied from NEB and synthesised
I-SceI homing endonuclease restriction site
Forward primer for sequencing/amplifying BioBrick parts (VF2)
Originally named VF2, primer binds upstream of the standard BBa_MCS on biobrick vectors.
A E. coli strain that carries the BsiWI gene from Bacillus species (D. Clark).
Andrew Wright, Dave Lane, Fran??ois-Xavier Barre, pDAG479 plasmid.
tetO Array Contains lots of tetO where bind YFP:tetR (<partinfo>BBa_K606025</partinfo>).
Reduce sequence to avoid restriction sites
Low to medium copy BioBrick standard vector
BBa_K592200 is a BioBrick standard vector with low to medium copy p15A replication origin (BBa_I50032) and spectinomycin antibiotic resistance marker, usable for Lambda Red recombineering in E coli. The E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
To make the plasmid usable for Lambda Red recombineering, all sequences with homologies to the E coli chromosome had to be removed. To accomplish this, the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
Erik Gullberg, Antonio Ascue Avalos, Erik Lundin
p15A low to medium copy replication origin BBa_I50032
SacII restriction enzyme site with extra flanking nucleotides
This is the scar produced by assembly using RFC 25. If you are assembling a composite part using RFC 25, you can insert this part and specify blunt assembly to get the desired sequence.
pRS316 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.
Has an RFC10 compatible MCS, with ADH Terminator. No further RFC10 restrictions sites in the backbone.
Ray Paul Laureano-Mercado & Mark Kirollos
This part is the coding sequence for the enzyme melamine deaminase, optimized for usage in E. coli. This enzyme eliminates an amine group from melamine to produce ammonia and ammeline and is able to remove another amine group at a rate 10 times slower than the first reaction to bring the compound to ammelide. The enzyme does not react to a measurable extent with this last compound, so it metabolizes melamine by consecutive and predictable deaminations.
The TriA gene???s codons were modified from the original and optimized for E. coli to a Codon Adaptation Index (CAI) of .76. After that, some palindromic and mRNA destabilizing sequences were removed to increase the stability of our DNA during synthesis.
The original gene coding for the protein of interest was isolated from a species of Pseudomonas (http://www.ncbi.nlm.nih.gov/nuccore/11890744).
Theophylline Responsive Riboswitch 1G1 with Engineered RBS+GFP generator
Theophylline riboswich is composed of a synthesized theophylline aptamer and a regulatory platform.
This is a GFP generator with a theophylline responsive riboswitch sequence. The mRNA with a theophylline riboswitch in it would respond to theophylline concerntration, producing different amount of GFP. Under the control of different promoters, it could be used to demonstrate the theophylline responsive curve, providing data for modeling. The theophylline riboswitch sequence could also be used to regulate the translation of other genes.
The RBS of this device is altered into AGGAGGU. When changing the RBS, the anti-RBS sequence should be changed as well.
Siyang HAO, Tong MU, Zheng LUO, Mengqi ZHANG
Theophylline responsive riboswitch Theo 1G1
This part is part of a family of Bioscaffold - Linkers. This part will help users interested in protein-fusion. The usage of the part will allow in frame assembly of 2(+) proteins by: *Removing the stop codons from the upstream protein. *Removing the scar created by the Assembly Standard 10 (RFC10) construction of 2(+) protein coding sequences. *Maintaining the start codon of the downstream protein. *Adding a flexible (Gly-Ser-Gly) linker between the two protein coding sequences. *Acting as a translational buffer if ligation is incorrect. ===Assembly Instructions=== *Create an Assembly Standard 10 fusion as below ; **Upstream Protein Part - Bioscaffold Linker - Downstream Protein Part** *Use the BioScaffold Specific Enzymes (i)Restrict with BpuEI to remove upstream scar (created between Upstream Protein - Bioscaffold) and stop codons from upstream protein part. (ii)Ligate and you will get the intermediate product; **Upstream Protein (w/out Stop codons) - Tyr - BioScaffold Linker - Downstream Protein** *Restrict with CspCI to remove downstream scar. *Ligate to get the final product; **Upstream Protein - Tyr-Gly-Ser-Gly- Downstream Protein** ====N.B.==== CspCI cuts with a 1 b.p discrepancy depending on the sequence. However this should not affect the restriction/ligation process as this has been taken into account and incorporated into the design. Even with the discrepancy involved the restriction/ligation process will still give the same product and in-frame fusion. The other restriction variants of CspCI have also been taken into account and relevant BioScaffolds are being produced to be tested. This version is for when CspCI cuts 13/10b.p. (top/bottom) on the 5' of the recognition sequence and 10/13bp (top/bottom) on the 3' of the recognition sequence.
The CspCI discrepancy had to be taken into account
HindIII restriction enzyme site with extra flanking nucleotides
HindIII restriction site with three flanking base pairs
NCBI database of Saccharomyces cerevisiae's genome
ori 7 region of Saccharomyces cerevisiae mtDNA, works as replication initial site.
This is a part consisting of only a single nucleotide ... T. It can act as a spacer between restriction sites and parts.
Overlap extension of synthetic oligonucleotides
DNA substrate for Tn5 transposase. Flanking a sequence with J61021 on the 5' end and J61022 on the 3' end makes it a functional transposon. pSB1A2-Bca1013
BamHI restriction site with extra flanking nucleotides
XbaI/SpeI mixed site. Simply used to aid in entry of parts into the registry.
3??? Integration Sequence for the amyE locus of B. subtilis
The 3??? integration sequence was taken from the shuttle vector pDR111 which has been used in many studies on B.subtilis, in particular in the studies of transcriptional control[1,2,3] References 1.Shimotsu H and Henner DJ. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene 1986; 43(1-2) 85-94. pmid:3019840. 2.Erwin KN, Nakano S, and Zuber P. Sulfate-dependent repression of genes that function in organosulfur metabolism in Bacillus subtilis requires Spx. J Bacteriol 2005 Jun; 187(12) 4042-9. doi:10.1128/JB.187.12.4042-4049.2005 pmid:15937167 3.Britton RA, Eichenberger P, Gonzalez-Pastor JE, Fawcett P, Monson R, Losick R, and Grossman AD. Genome-wide analysis of the stationary-phase sigma factor (sigma-H) regulon of Bacillus subtilis. J Bacteriol 2002 Sep; 184(17) 4881-90. pmid:12169614
Integration sequences allow DNA to be incorporated into the chromosome of a host cell at a specific locus using leading (5') and trailing (3') DNA sequences that are the same as those at a specific locus of the chromosome. The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143002) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The AmyE locus was the first locus used for integration into B.subtilis by Shimotsu and Henner[1] and is still commonly used in vectors such as pDR111[2], pDL[3] and their derivatives. Integration at the AmyE locus removes the ability of B.subtilis to break down starch, which can be assayed with iodine as described by Cutting and Vander-horn[4]. The 5' and 3' integration sequences for the AmyE locus were used to integrate the Imperial 2008 iGEM project primary construct into the B.sutbilis chromosome. References 1. Shimotsu H and Henner DJ. Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene 1986; 43(1-2) 85-94. pmid:3019840 2.Nakano S, Kster-Schck E, Grossman AD, and Zuber P. Spx-dependent global transcriptional control is induced by thiol-specific oxidative stress in Bacillus subtilis. Proc Natl Acad Sci U S A 2003 Nov 11; 100(23) 13603-8. doi:10.1073/pnas.2235180100 pmid:14597697 3.Bacillus Genetic Stock Center; www.bgsc.org 4.Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27???74.
The AmyE integration sequence was taken from the vector after comparison by BLAST to the B.subtilis chromosome to identify the homologous sequences. The sequence present in both the host chromosome and the plasmid at the 3' end of the gene is the 3' sequence required for integration
Right end of a linear transpososome sequence containing the 19 bp mosaic end in the reverse orientation. The sequence also contains a PvuII cut site which releases the transpososome when cut with PvuII. Constructed compound parts intended for transposition must not contain other PvuII end, or be PCR amplified with blunt end producing enzymes (Pfu, Phusion) at the PvuII cut site between CAG and CTG. This part, and the companion part M1000 provide Biobrick tools to construct Tn5 transpososomes: linear DNA sequences which can be cut in vitro from a plasmid backbone, or created by PCR, combined in vitro with purified Tn5 trasposase protein, electroportated into cells, and finally randomly inserting into the bacterial chromosome. The parts assembled between the two mosaic ends can be delivered, including antibiotic resistance markers or other functional chromosomal insertions.
Synthesis from overlapped oligos with PCR.
Contains both a BbsI and a BstXI restriction site. When cut with both enzymes, it leaves an EcoRI and a PstI site.
70 bp from Eco to Spe or Xba to Pst, to allow for gel extraction. The middle portion is largely noise, intended as filler to raise the size of the part.
SapI restriction enzyme site, reversed (offset cutter)
SapI restriction site in the reverse orientation (recognition sequence on the 3' strand). SapI is an offsite cutter. From left to right this part includes the designed 3 bp 5' overhang (ATG), a single bp spacer, the 7 bp recognition sequence (5'-GCTCTTCT-3'), and two bp spacer. Cleavage at this site results in a 3 bp, 5' overhang (left side, top strand), which makes it compatible with Heather Keller's RBS assembly scheme for creating RBS/CDS junctions without a mixed site. This part is specific for CDSs with an ATG start site and is not compatible with GTG start sites (via HKs assembly scheme). The single bp spacer on the 3' end allows RBSes construct with this part to be used in standard biobricks assembly and is necessary to maintain the reading frame of a downstream CDS. Note that Standard Biobricks Assembly with this site results in the creation of a new ATG start site, and a 7 amino acid fusion at the N terminus of the downstream protein.
This part only exists in composite parts within the registry. This restriction site is used by Heather Keller for creating blunt junctions between RBS sequences and coding regions (i.e. no biobricks scar in between).
This is a non-coding RNA that encodes the pyrroloquinoline quinone binding aptamer. It can be used in both binding and sensing applications.
This was one of the best two DNA aptamer candidates from SELEX (also see BBa_K2027049).
Scar site created by A part ligation. Used for entering composite parts on the Registry.
This part is the standard BioBricks suffix.
All Oligos for Tetrahedral Nanostructure
This will be used to build and express our desired functional nanostructure in E. coli that target micro RNA biomarkers. This plasmid encodes all the ssDNA that will make the tetrahedral nanostructure, i.e. Oligos 1, 2, 3, 4 and 5.
Reverse BioBrick primer annealing site (VR binding site)
This is the VR sequence to be used in construction of plasmids which include the VR primer site. It is the reverse complement of BBa_G00101.
Oligo 5 of DNA Tetrahedral Nanostructure
This will be used to build our desired nanostructure that target micro RNA biomarkers. This plasmid encodes the second ssDNA that will make the tetrahedral nanostructure, together with oligo 1, 2, 3 and 4.
XmaI restriction enzyme site with extra flanking nucleotides
XmaI restriction site with 3 flanking base pairs
This sequence is produced by an assembly method.
This sequence is produced by RFC10-assembly of a RFC25-CDS with a RFC10-prefixed part.
The resulting sequence is a combination of 9 nucleotides from the RFC25 prefix (acc ggt taa) and the RFC10 scar (tactagag).
DNA substrate for Tn5 transposase. Flanking a sequence with J61021 on the 5' end and J61022 on the 3' end makes it a functional transposon.
A Composite BioScaffold Part (PpiI and PsrI) for Protein Fusions (see Design Page)
Brings together a BioScaffold part that removes protein tails with one that removes protein heads in order to create a BioScaffold part that can help create protein fusions using standard assembly
The part that links the two BioScaffold parts can be altered as desired--here I just wanted to leave at least 7 bp beyond the internal scars.
This part comes from http://www.ncbi.nlm.nih.gov/nuccore/70907607?from=48797&to=51040&report=gbwithparts and corresponds to range 48797..51040 of the R751 sequence.
The relaxase / helicase TraI of the IncP beta plasmid R751. Essential of conjugative transfer. This part nicks oriT. It should be placed on a message plasmid along with oriT, and will enable transfer of the message plasmid in the presence of a modified R751 helper plasmid such as BBa_I714037.
Right Library Insert Preparation Part (To use with J70050, and J70040)
Still in Testing Surround a part with J70050 and J70052, digest with AarI Insert into a vector that contained J70040 and had J70040 removed by AarI and MabI
Not sure if one AarI site on each site will be enough.
We use this sequence to insert into staples as a level 1 pixel.
Rothemund PW. Folding DNA to create nanoscale shapes and patterns. Nature. 2006 Mar 16;440(7082):297-302.
S50 is the streptavidin-binding component of the A50 adaptamer which can physically link together the proteins thrombin and streptavidin. A50 contains a 45 bp double-stranded linking region between thrombin and streptavidin aptamer sequences.
http://openwetware.org/wiki/Silver:_Oligonucleotide_Inserts Oligonucleotide Inserts
BglII and BamHI restriction sites with some flanking
5??? Integration Sequence for the amyE locus of B. subtilis
The 5??? integration sequence was taken from the shuttle vector pDR111 which has been used in many studies on ''B.subtilis'', in particular in the studies of transcriptional control#1 #2 #3 <biblio> #1 pmid=14597697 #2 pmid=15937167 #3 pmid=12169614 </biblio>
The 5' integration sequence can be added to the front of a Biobrick construct and the 3' integration sequence specific for this locus (Part BBa_K143002) to the rear of the Biobrick construct to allow integration of the Biobrick construct into the chromosome of the gram positive bacterium B.subtilis. The AmyE locus was the first locus used for integration into ''B.subtilis'' by Shimotsu and Henner#1 and is still commonly used in vectors such as pDR111#2, pDL#3 and their derivatives. Integration at the AmyE locus removes the ability of ''B.subtilis'' to break down starch, which can be assayed with iodine as described by Cutting and Vander-horn#4. The 5' and 3' integration sequences for the AmyE locus were used to integrate the Imperial 2008 iGEM project primary construct into the ''B.sutbilis'' chromosome. <biblio> #1 pmid=3019840 #2 pmid=14597697 #3 ''Bacillus'' Genetic Stock Center [www.bgsc.org] #4 Cutting, S M.; Vander-Horn, P B. Genetic analysis. In: Harwood C R, Cutting S M. , editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons, Ltd.; 1990. pp. 27???74. </biblio>
The AmyE integration sequence was taken from the vector after comparison by BLAST to the ''B.subtilis'' chromosome to identify the homologous sequences. The sequence present in both the host chromosome and the plasmid at the 5' end of the gene is the 5' sequence required for integration.
software generated kanamycin aptamer (candidate 1)
Bacillus stearothermophilus GR75 (Z. Chen)
This sequence is produced by RFC10-assembly of a RFC10-RBS with a RFC25-prefixed part.
The resulting sequence is a combination of the RFC10 RBS-CDS scar (tactag) and 9 nucleotides from the RFC25 prefix (atg gcc ggc).
Oligo 4 of DNA Tetrahedral Nanostructure
This will be used to build our desired nanostructure that target micro RNA biomarkers. This plasmid encodes the second ssDNA that will make the tetrahedral nanostructure, together with oligo 1, 2, 3 and 5.
This sequence does not come from a genomic sequence. First reported by Li et al 2010, it is comprised by 16 nucleotides with a high percentage of G which are the ones capable of stabilising and forming the quadruplex with the lead ion.
This aptameric sequence is capable of recognising and binding specifically to lead II ions. This sequence, which works as a single stranded nucleic acid molecule, is capable of forming a G-quadruplex by interacting with lead ions (Pb2+) while disrupting a partially complementary duplex.
For standardisation purposes and its usage in iGEM, this sequence was cloned as a duplex on pSB1C3 thereby adding the prefix and suffix sequences to it. Given the fact that the sequence is need as a single-strand molecule, a primer was designed in order to carry out a PCR with a single primer for the production of this molecule.
Roger Milton Rubio Sanchez, Ana Laura Torres
This part contains the WT loxP site, which is recognised by Cre recombinase (Part BBa_K1680007), in forward orientation.
Improved insertion site with two RNAse E cut sites
Shortened RNAse E site, insertion site, duplicate RNAse E site, and a trailing 5' hairpin (HP16 from Smolke et al., 2002).
This artificial DNA sequence is converted from text with the use of ASCII system. The specific repeat sequence "repeat 1" and "repeat 2" are come from transfer region in R64 plasmid, with genomic assession no.:AB027308.1
This message system consists of a unique designated message sequence, being inserted with specific repeat sequence for recognition site by Rci site-specific recombinase.
This DNA sequence is acting as a template, to guide the make of a message inserted with specific repeat sequence for recognition by Rci recombinase in order to accomplish random rearrangement of message in encryption process.
Left Library Insert Preparation Part (To use with J70052, and J70040)
Still in Testing Surround a part with J70050 and J70052, digest with AarI Insert into a vector that contained J70040, and had J70040 removed by AarI and MabI
Not sure if single AarI site will be good enough
OriTf (Origin of Transfer for the F-type conjugative plasmid) is the nic region, is where the relaxosome nicks the plasmid and conjugative transfer by F plasmid machinery begins via rolling circle replication. This particular plasmid has been derived from a F-type plasmid donated by Professor Laura Frost University of Alberta. NOTE: OriTf is NOT the same as the host cell's native Origin of Transfer (this is a separate site within the bacterial host's main genome).
Conversion of the iGEM parts registry to SBOL2.1