Transfection vector (similar to pSB1AK8)
Low-copy plasmid pTRC99A (trc promoter and lacI removed)
PRS306 is a shuttle plasmid which can express great both in E.coli and yeast.It has Amp resistance and it is URA auxotroph.
It is a popular shuttle plasmid in yeast.
pSB4C5 with RBS-RFP-TT between SpeI-PstI
When the SpeI-PstI DNA fragment from BBa_J61002 , containing the mRFP1 gene with a strong RBS and a double terminator, is transferred into pSB4C5 (digested SpeI-PstI) the resulting plasmid is BBa_J107055 . See pSB4C5 for detailed feature information.
This is not a standard BioBrick plasmid, as the suffix is not SpeI-NotI-PstI.
pSB3K3 (backbone) and BBa_J61002 (SpeI-PstI insert).
Commercial plasmid used to make BBa_K209432
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).
All forbidden restriction sites have been removed from the plasmid backbone, making it RFC25 compatible .
Based on invitrogen's pYES2 plasmid. For more information see the part information of pTUM100 (BBa_K801000).
This is a Low Copy BioBrick 2.0 Plasmid with the Ampicillin Resistance Gene ampR.
This is modified and converted from the BioBrick 1.0 Plasmid pSB4A3.
beta-lactamase ampicillin resistance gene
The forth vector of the Golden gate series (adapted from psb1c3) This vector expedites golden gate cloning with BBa_K560012, BBa_K560013 and BBa_K560014. To make this set of vector compatible with the standard biobricks, we have maintained PstI and XbaI recognition sites. Flanking these two sites are the cleavage and recognition sites of BsaI, a kind of Type IIS restriction endonuclease. This kind of restriction endonucleases cleaves outside their recognition sites thus creating different sticky ends. If you want to assemble four biobricks at a time, you can put the biobricks in order into corresponding vector by the digestion of PstI and Xbal. Then mix these assembled vectors and a backbone (PFUS4 from TALEN golden gate kit). Under the effect of ligase and Bsal, these biobricks will have sticky ends nose to tails and ligate in order in a single reaction.
Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research, 2011, Vol. 39, No. 12.
ampicillin resistant split R6K vector w/ O16 HAs and FRT-Kan-FRT
ampicillin resistant split R6K vector with O16 homology and kanamycin resistance flanked by FRT sites. Requires expression of pir to replicate. Referred to internally as pBjk2648r
pSBBs1C: Empty backbone for integration into Bacillus subtilis amyE locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the amyE locus and can be selected with Chloramphenicol (cat gene). It has a ampicillin resistance for cloning in E.coli. It originates from the Bacillus vector pDG1662. This BioBrick is part of the LMU 2012 Bacillus BioBrick Box project: http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR.
This backbone is an igem compatible version of the Bacillus vector pDG1662
pBA Universal Plasmid for BioBytes Gene Assembly System
The boundary sequences required for sticky end production for the BioBytes gene assembly system are acquired by inserting gene sequences destined to become bytes into either of the host plasmids pAB or pBA. Both are derivatives of pUC19 with the following key modifications: 1. Replacement of the pUC19 MCS with the MCS shown in figure 1. 2. Elimination of the Z alpha fragment 3. Elimination of the restriction sites Bts1 and BspQ1 Functional features include: 1. The ability to adapt biobricks to the Byte format using the Xba1 and Pst1 sites contained in the MCS. 2. The ability to adapt the in the ASKA collection (Kitagawa 2005) to the Byte format (4000+ E. coli K12 ORFs). ORFs are cloned into an expression vector, accompanied by a 6xHis tag at the N-terminus and a GFP fusion on the C-terminus. ASKA parts can be freed by digestion with SfiI, leaving noncomplementary 3 bp 3' sticky ends, which can be cloned directionally into the MCS after digestion with BstAPI. Since the ASKA parts are freed from the interior of the CDS, the MCS provides a start codon 10 bp downstream of the RBS, and a stop codon (TGA) downstream of the insertion. ORFs cloned from ASKA plasmids replace the N-terminal methionine of the predicted ORF with Met-Ala-Leu-Arg-Ala, and append Cys-Leu at the C-terminus. 3. Other potentially useful restriction sites have been included in the MCS as well as an RBS consensus positioned 8 bp upstream of the ATG used for ORF???s derived from 1. And 2 4. The ability to select for insert-containing constructs through the linearization of parental contaminants by cleavage with the rare- cutters SwaI and PmeI contained in the MCS. 5. Two routes for the production of 12 base overhangs by PCR using universal primers: A) PCR followed by nicking using the engineered enzymes Nb.BbvCI and Nb.BtsI. B) PCR followed by removal of a primer-encoded uracil residue using BioLab???s UserTM system. Primer positions and sequences are indicated for both.
Using these plasmids as well as universal primer sequences for the PCR extraction of bricks from the plasmids anyone will be able to use this system for their own rapid assembly. The design ensures that the existing catalog of BioBrick parts is compatible with the system, and can be easily cloned into the universal plasmids for brick generation. We believe that this new system is ready for both use and formalization, and we have prepared a BBF RFC detailing the use of the BioBytes system
Insulated low copy vector based on pSB1A2. Uses B1010 transcriptional terminator upstream of BBa prefix and B0025 double transcription terminator downstream of BBa suffix. Uses I50042 low copy pSC101 replication origin.
Contains stuffer piece of GCATGCATGCAT to be removed when cloning parts.
Insulated vector based on pSB1A2. Uses B1010 transcriptional terminator upstream of BBa prefix and B0025 double transcription terminator downstream of BBa suffix.
Antibiotic-free selection plasmid (pSB1C3_alr_RFP)
Verification reverse (VR) primer binding site
Verification forward (VF2) primer binding site
It's the same plasmid backbone of pSB1A3, but sequence between SpeI and PstI in the suffix are from plasmid backbon J61002. SpeI-RBS-mRFP-Terminator-PstI-pSB1A3-prefix
This plasmid backbone is generated by cutting a part in J61002 with X+P, and ligating it into pSB1A3 cutted with X+P.
kanamycin resistant p15A vector. referred to internally as pBjo1601K
Plasmid backbone pSB1A3 carrying the lac operon induction system
This plasmid is derived originally from pBeloBAC11 with multiple modifications to make it compatible with biobricks and more useable. Without modification, J61031 is a high-copy plasmid in normal laboratory strains of E. coli due to the colE1 origin of replication located within the biobrick part region. It also confers ampicillin resistance, kanamycin resistance, and constitutively-expressed RFP. Upon replacement of the SpeI/XbaI region, the colE1, ampR, and RFP features are removed, leaving behind the F plasmid replicon, the KanR gene, and an R6K origin of replication. At this point, the plasmid will replicate as a medium or high copy plasmid in pir+ or pir116 strains such as Ec100D::pir116. In non-pir strains (most lab strains), the plasmid will replicate at near-single copy levels. The restriction sites EcoRI, SpeI, NotI, and XbaI are all unique in this plasmid. There are several PstI sites, however. pBACr-NESd
kanamycin/neomycin resistance marker (NTP II marker)
beta-lactamase ampicillin resistance marker
finO incD/sopC region; contains 12 43 bp direct repeats that bind SopB protein; required for partitioning of F plasmid to daughter cells after replication as well as repression of sopAB operator;
Part-making plasmid with Ptet-XbaI-[biobrick]-SpeI-PstI
Plasmid for constructing biobrick parts that provides a minimal set of functions to observe activity.
pSB1A2-I13521 See KAD062806-Construction of J23006 for details
Gateway entry vector for promoters (L4R1)
Laura Deming, Joy Jiao, Adrian Slusarczyk
This plasmid is PGAPzα C + modified Multiple Cloning Site(MCS) (Linker) fixed according to iGEM standards. We replaced old multiple cloning cite with new modified multiple cloning site, which has new cutting sites according to iGEM standard. The original MCS has ECORI, KpnI, NotI and XbaI restriction enzyme binding sites compared to the new MCS we designed in which there are ECORI, NotI, XbaI, SpeI and PstI restriction enzyme cutting sites. PGAPzα C + Linker has α-secretion factor before MCS which allows yeast cells and some Eukaryotic cells to secrete the protein into the outside of the cell membrane. Also after MCS there are MYC epitope and 6x histag that are used to tag the protein so it can be easily identified by westernblot technique.
We replaced old multiple cloning cite with new modified multiple cloning site, which has new cutting sites according to iGEM standard.
Plasmid Backbone for golden gate assembly
This backbone is suitable for golden gate assembly. Golden gate assembly is a suitable assembly to combine many parts in order you want. This plasmid backbone have restriction enzyme cutting site of Dpn1 let you build construction by Golden gate assembly in one ligation.
The terminal of this part was modified to use it for Golden gate assembly
This part was made by PCR from iGEM part BBa_PSB1K3
This is a high copy plasmid for use in assembly of BioBrick® standard biological parts.
This part was derived from pSB1AC3-P1010.
Variant of pBca1021 (J61003) with GFP inserted. In practice, it is a E0040 basic part just in a different pSB1A* plasmid.
beta-lactamase (ampicillin resistance marker)
plasmid vector for Rhodobacter sphaeroides
This vector is based on pMG170/pMG171. LacZ promoter and lac reporter have been removed from original vector.
To make this vector more flexible and easier to control, we have deleted the lacZ promoter and lac reporter and made it a standard BioBrick vector.
Vector with pSS Prefix A and Suffix B, confers kanamycin resistance, p15a origin of replication, constitutively expresses luxR and araC.
Inserted luxR and araC to be constitutively expressed in the reverse direction on the vector. Removed restriction sites in prefix and suffix to improve assembly success rate.
Modified pGA3K3 from 2011 University of Washington iGEM team.
pORE Gus Reporter Series Vector with BiOBrick MCS (Kan resistance)
Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Basta resistance in plants. Has a constitutive plant promoter and in-frame Gus reporter. Conforms to Silver BioBrick Assembly Standard 21.
Modified vector pORE_R from the Arabidopsis Information Resource
pSB3K3 with RBS-RFP-TT between SpeI-PstI
When the SpeI-PstI DNA fragment from BBa_J61002 , containing the mRFP1 gene with a strong RBS and a double terminator, is transferred into pSB3K3 the resulting plasmid is BBa_J107055 . See pSB3K3 for detailed feature information.
Commercial plasmid used to make part BBa_K209473.
pBS0KPspac, an IPTG-inducible replicative expression vector for
pBS0KPspac, an IPTG-inducible replicative expression vector for ''Bacillus subtilis''.
Inducible expression vector with RFP-MCS downstream of Pspac.
This vector is derived from pDG148, and the "repaired" version of pSBBs0K-Pspac.
pSB3C5 + MID and AF TALEN (K1480001) Target sites
Backbone vector of pSB1C3 that contains two modifications with a TALEN target site on the center and after the origin of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains two TALEN target sites (MID+AF)
Modifications where inserted by PCR on both directions due to TALEN specificity.
Plasmid pGAPzalpha A standardized according to iGEM standards, with RFP sequence.
kanamycin resistant split R6K vector. Requires pir expression to replicate. referred to internally as pBjk2164
Low copy BioBrick temperature sensitive standard vector
pSB8A15 is a BioBrick standard vector with low copy pSC101ts replication origin (~5 copies per cell) and ampicillin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 repelication origin and propagates at 30???C but stop replicating at 42???C. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22
The resistance cassette is terminated by a Lux bidirectional terminator. Adjacent to this terminator there is a designed primer binding site to where the forward primer for lambda red can be designed to bind to no matter what the insert in the cloning site is or what the resistance is. The resistance cassette is flanked by SalI and SacI restriction sites for easy switching of resistance in the backbone. The pSC101ts origin of replication is flaked by NheI and MluI restriction sites for easy switching of origin of replication.
Vector with pSS Prefix A and Suffix D, confers chloramphenicol resistance, p15a origin of replication, constitutively expresses luxR and araC.
This integrative plasmid facilitates transformation of Synechocystis PCC6803 and similar species. It inserts gene of choice into the Synechocystis cpc gene by homologous recombination.
Primers containing biobrick prefix and suffix as well and other described parts were used to make this backbone RFC10 compatible.
Plasmid was purchased from open source plasmid repository Addgene.com (originally named cpc-PHLS (g)). It was modified to be biobrick compatible. Formighieri, C., & Melis, A. (2014). Regulation of β-phellandrene synthase gene expression, recombinant protein accumulation, and monoterpene hydrocarbons production in Synechocystis transformants. Planta, 240(2), 309-324.
Low copy BioBrick standard vector with LacI
pSB4C15Iq(FRT) is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and chloramphenicol antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. This vector carries a LacIq cassette on the backbone for tight repression of any lac promoter cloned into the cloning site.
The resistance cassette is terminated by a Lux bidirectional terminator. Adjacent to this terminator there is a designed primer binding site to where the forward primer for lambda red can be designed to bind to no matter what the insert in the cloning site is or what the resistance is. The resistance cassette is flanked by SalI and SacI restriction sites for easy switching of resistance in the backbone.
This plasmid was derived from pSB1AK3-P1010.
pGEM-T-easy Vector is linearized vector with a single 3??-terminal thymidine at both ends. The T-overhangs at the insertion site greatly improve the efficiency of ligation of PCR products by preventing recircularization of the vector and providing a compatible overhang for PCR products generated by certain thermostable polymerases. pGEM-T-easy Vector is high-copy-number vector containing T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the α-peptide coding region of the enzyme β-galactosidase. Insertional inactivation of the α-peptide allows identification of recombinants by blue/white screening on indicator plates. Choice of Restriction Sites for Release of Insert: pGEM-T Easy Vector contains numerous restriction sites within the multiple cloning region. Its multiple cloning region is flanked by recognition sites for the restriction enzymes EcoRI, BstZI and NotI, providing three single-enzyme digestions for release of the insert. Alternatively, a double-digestion may be used to release the insert from either vector. pGEM-T Easy Vector System is supplied with 2X Rapid Ligation Buffer. Ligation reactions using this buffer may be incubated for 1 hour at room temperature. The incubation period may be extended to increase the number of colonies after transformation. Generally, an overnight incubation at 4??C produces the maximum number of transformants.
This gene was designed by Promega. We take no credit. It has been sponsored by Bioneer.
SP6 RNA polymerase transcription initiation site
pUC/M13 Reverse Sequencing Primer binding site
T7 RNA polymerase transcription initiation site
pUC/M13 Forward Sequencing Primer binding site
pSB1C3 + MID and BF TALEN (K1480001) Target sites
Backbone vector of pSB1C3 that contains two modifications with a TALEN target site on the center and before the origin of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains two TALEN target sites (MID+BF)
pSB4A15Iq(FRT) is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and ampicillin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. This vector carries a LacIq cassette on the backbone for tight repression of any lac promoter cloned into the cloning site.
from the addGene plasmid https://www.addgene.org/66843/
Integration vector for Bacillus subtilis derived from pSac-Cm
This plasmid was derived from pSac-Cm by insertion of biobrick compatible restriction sites (prefixes and suffixes), a terminator (BBa_B0015) after the suffixes sequences, and red fluorescent protein sequence (RFP) in between the prefix and suffix in its multiple cloning sites (MCS). New biobricks can be inserted into this vector by replacement of the RFP biobrick, and selection of the white colonies. This backbone has a multi host replication origin and replicates in E. coli. The plasmid is designed to integrate a cloned insert into the B. subtilis chromosome via double recombination between plasmid and chromosomal sacA sequences, and in order to check for integration a simple Phenol Red Sucrose test can be perform, if sacA is no longer functional, they will remain ph7, if they are still able to digest sucrose their pH will be under 5.
We found that that the integration plasmid that groningen send to the registry were unable to integrate in B. subtilis. This is due to the high number of mutations in the sacA integration region.
Plasmid for Plug'n'Play. Contains an Ampicillin resistance marker.
Compatible with the Plug'n'Play assembly standard.
This is the plasmid backbone of an expression vector after performing an LR reaction in Gateway cloning assembly. It contains attB and attP sites that are the result of the recombination of attL and attR. The selection marker for this expression vector is ampicillin.
pCK01 converted into BioBrick vector (formerly BBa_I739202)
Plasmide with a Ampicilin selecting marker and Multiple cloning site within the lacZ(selection marker) gene on it.
quickly selecting plasmide with lacZ marker on it.
pSB1C30: High copy BioBrick cloning/expression backbone carrying Cm resitance
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena B??scher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Sch??fer, Carolin Schmelas, Silvan Schmitz, Max Waldha
Was archived by cloning an oligo containing the promoter into the EcoRI site of pSB1C3
pSB4T3 is a low copy plasmid with tetracycline resistance. It has a repA pSC101-derived replication origin. (Copy number ~10-12, Lutz and Bujard, 1997.) It is constructed from the BioBricks vector scaffold BBa_I51001.
See http://openwetware.org/wiki/Synthetic_Biology:Vectors/pSB%2A%2A5_design for more information.
BBa_I51001 vector scaffold, BBa_I50040 replication origin and BBa_P1005 resistance marker.
This part is a biobrick backbone with chloramphenicol resistance and pBR322 derived origin. The copy number is expected to be around 20. The characterization of this part is currently happening, and more data will be uploaded soon. Note: The backbone contains an extra VR sequencing site from the origin K415039. This site will be removed in the final version pSB6C5. Also, cells containing this plasmid grow slower than usual.
The extra reverse sequencing site needs to be removed in the final version (pSB6C5). The resistance P1004 was placed before the origin operon K415039 to provide a promoter to the copy-number regulating protein ROP.
This backbone was constructed with the standard biobrick plasmid construction method. The origin is K415039 and the chloramphenicol resistance is from P1004. After several failures in getting the correct colonies with the ccdB containing base vector I51020, the appropriate base vector region was obtained by PCR of an existing version 5 backbone containing RFP (pSB3T5). The RFP version of the base vector allowed for easy screening for correct colonies.
Low to medium copy BioBrick standard vector
pSB3K3-1 is a medium copy plasmid with kanamycin resistance. It has a p15A pMR101-derived replication origin, copy number 20-30. (Lutz and Bujard, 1997) pSB3K3-1 has a terminator upstream of its MCS, which is oriented to prevent transcription from *inside* the MCS from reading out into the vectorl. A second terminator (E.coli His operon-derived) is downstream of the MCS, again insulating the vector from transcription reading out of the MCS. Ideally, future versions of standard biobrick vectors would have terminators bracketing the MCS that were 100% efficient in terminating transcription both into and out of the MCS region.
pAB Universal Plasmid for BioBytes Gene Assembly System
Using these plasmids as well as universal primer sequences for the PCR extraction of bricks from the plasmids anyone will be able to use this system for their own rapid assembly. The design ensures that the existing catalog of BioBrick parts is compatible with the system, and can be easily cloned into the universal plasmids for brick generation. We believe that this new system is ready for both use and formalization, and we have prepared a BBF RFC detailing the use of BioBytes.
Multi-host replicative plasmid, for B. Subtilis and E. Coli
This plasmid is an multihost plasmid, replicated both in E. Coli at high copy number (>200) and low copy in B. Subtilis (about 20). This plasmid is ideal for importing parts in Subtilis: all the cloning can be done in E. Coli, as well as the amplification. Once the plasmid is extracted, you can incorporate it directly in B. Subtilis using electroporation. This plasmid is Ampicilyn resistant in E. Coli, and is tetracycline resistant in both B. Subtilis and E. Coli. The biobrick BBa_J04450 is inserted between the E X and SP site, has a negative cloning control in E. Coli
The EcoRI site has to be eliminated and the Biobricks prefixes and suffixes were recovered
The plasmid was obtained from Harald Putzer laboratory at the IBPC [http://www.ibpc.fr/UPR9073/equipe_HaraldJackie/AccueilJAP_HPGB.htm] . This is the fusion of the commercial plasmids pBC16-1 and pTZ19R via the site EcoRI. This site has been eliminated by the 2011 iGEM Paris Team by quick change mutagenesis, and the multicloning site was transformed into the biobrick standards.
The Potsdam Assembly Standard is a modified PLICing (Phosphorothioate-based ligase-independent gene cloning) method ([http://www.ncbi.nlm.nih.gov/pubmed/20646988 Blanusa ''et al.'' (2010)]). For this standard we designed a new cloning backbone with an RFP expression cassette as insert and two new restriction enzyme recognition sites in the suffix and prefix in pSB1C3, the Potsdam Standard Backbone. In the prefix, we added the Apa I recognition site and in the suffix the Sph I recognition site. Both enzymes causing a 3??? overhang with 4 nucleotides. For the cloning process we cut the vector with Apa I and Sph I. In this assembly standard that???s the only step where we have to use restriction enzymes. The gene of interest was amplified with primers that contain 4 phosphothioate nucleotides and the recognition sites for Apa I and Sph I at the 5??? end. After incubation in an iodine/ethanol solution, the thiophosphates were cut out resulting in a 3??? overhang which is suitable to the overhangs that was created by cutting the new assembly vector. After that the digested vector and the PLICed insert were mixed and transformed into E. coli. By using the RFP expression cassette we created a ligation control system. Due to the fact that red fluorescent colonies have a failed vector ligation we can tell which colonies are correctly ligated. The colonies that do not show red fluorescence are the positive clones.
To create the Potsdam Standard Backbone, we amplified the RFP expression cassette (BBa_J04450) to add the restriction site for Apa I and Sph I. Then, the amplified expression cassette was insert into pSB1C3 using Xba I and Pst I.
pGA4A5, Gibson assembly plasmid (bglBrick) with pLac-GFP insert
Michael Brasino, Alicia Wong, Rashmi Ravichandran
Backbone that is Gibson reaction friendly (Bgl Brick)
This plasmid is PGAPzα C + modified Multiple Cloning Site(MCS) (Linker) fixed according to iGEM standards. PGAPzα C + Linker has α-secretion factor before MCS which allows yeast cells and some Eukaryotic cells to secrete the protein into the outside of the cell membrane. Also after MCS there are MYC epitope and 6x histag that are used to tag the protein so it can be easily identified by westernblot technique.
We replaced old multiple cloning cite with new modified multiple cloning site, which has new cutting sites according to iGEM standard. The original MCS has ECORI, KpnI, NotI and XbaI restriction enzyme binding sites compared to the new MCS we designed in which there are ECORI, NotI, XbaI, SpeI and PstI restriction enzyme cutting sites.
This plasmid backbone is the same as BBa_K245004 except that the restriction sites AgeI and XmaI are exchanged (which results in NgoMIV and XmaI restriction sites in prefix AgeI and BspEI in suffix). This detail enables extension of part cloned into vector with SG on C-terminal or/and PG on N-terminal.
ccdB was multiplied from BB-NIC-II-HisN (BBa_K245004) by PCR using primers which contained restriction sites in different combinations than those found in BB-NIC-II-HisN next to ccdB. Fragment was then ligated in BB-NIC-II-HisN cut with NgoMIV and SpeI.
The basis for BB-NIC-III-HisN is vector pSB1A2. Modified prefix and suffix are of synthetic origin. ccdB originates from part BBa_K245004.
pAUR135 RFC25 is a modified shuttle vector including DNA sequence to remove the vector region containing selectable marker AUR1-C from transformants of S.cerevisiae by this vector which allows repeatedly transformation. With optimization, K1641999 can be applied on RFC standard.
Commercial plasmid used in the construction of other composite parts.
pSB1C3 yGG transcriptional terminator acceptor vector
The original psb1c3 backbone was modified in order to allow for Golden Gate enabled parts to easily become biobricks.
Igem Registry of Standard Biological Parts.
pSB4A15Iq is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and ampicillin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. This vector carries a LacIq cassette on the backbone for tight repression of any lac promoter cloned into the cloning site.
pSB8C15 is a BioBrick standard vector with low copy pSC101ts replication origin (~5 copies per cell) and Chloramphenicol antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 repelication origin and propagates at 30???C but stop replicating at 42???C. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
Requires pir gene provided in trans to replicate.
Vector pYD, and can transfer into nucles of S.cerevisivae
pYD is a 5.0kb expression vector designed for expression, secretion, and display of proteins on the extracellular surface of S.cerevisivae cells. Features of this vector allow regulated expression, secretion and detection of expressed proteins.
In our project, to make our mutation of Tor2 easier, we separate complete Tor2 into three parts and mutate them independently. So we reconstruct pYD vector to add certain restriction sites for linking mutant Tor2 and original Tor2
AGA2, GAL1, TRP5 and CEN come from S.cerevisivae. Amppicillin resistance gene and the pUC origin for selection and replication come from E.coli
This plasmid backbone is based on pMA from Geneart. It has BioBrick prefix and suffix flanked on both sides by 50 bp of homology arms for insertion of the contained BioBrick into the 05000 locus of pCC2FosMB (BBa_K260001) by recombineering. The pMA-@CC2FosMB-05000bp backbone is available with BBa_K260015, which is composed of promoter-FRT-dhfr. If the PvuII fragment from BBa_K260007-BBa_K260015 is transferred by recombineering to the the 05000 locus of BBa_K260001-BBa_K260016, the intervening sequence between the FRT sites of BBa_K260015 and BBa_K260016 on the BBa_K260001 backbone will be 5,000 bp exactly. These plasmid backbones are similar and all allow recombineering-mediated transfer of the contained BioBrick (typically BBa_K260015) to the pCC2FosMB backbone BBa_K2600001. They differ in the homology arms, which determines the locus of insertion into pCC2FosMB. The bp numbers denote final inter-FRT distance as described above: BBa_K260004: pMA-CC2FosMB-00500bp BBa_K260005: pMA-CC2FosMB-01000bp BBa_K260006: pMA-CC2FosMB-02000bp BBa_K260007: pMA-CC2FosMB-05000bp BBa_K260008: pMA-RQ-CC2FosMB-10000bp
The 50 bp homologies were designed to delete 100 bp of BBa_K260001-BBa_K260016 and put the FRT site of BBa_K260015 (if used with the present backbone BBa_K260007) at a defined distance from the FRT site of BBa_K260016.
Geneart plasmid pMA, with 2x 50 bp from pCC2FosMB K260001.
BBa_K260001 (pCC2FosMB) 3' homology FRT-05000bp
BBa_K260001 (pCC2FosMB) 5' homology FRT-05000bp
This part is similar to part BBa_K1138001 with the difference that an RBS sequence is inserted between the XbaI and NsiI restriction sites. This part can serve as a control while doing experiments utilizing part BBa_K1138001. This plasmid consists of the mcherry gene (encoding red fluorescence protein) inserted in the backbone of pAW50 vector. EcoRI-NotI-XbaI-RBS-NsiI restriction sites have been introduced followed by a linker sequence followed by the mcherry sequence. The use of these sites will allow users to introduce any desired gene of their choice to be fused with the linker which is already fused with the mcherry sequence. This way any protein can be qualitatively studied in Methanogens via red fluorescence. Note:- the users will have to introduce the RBS sequence and start codon, delete the terminator sequence of their gene while cloning.
Primers were designed in a way to introduce EcoRI-NotI-XbaI-RBS- NsiI sites before the linker.
low to medium copy standard biobrick plasmid backbone
psb4A5, but ORI exchanged with ORI of pSEVA 171
Plasmid with a P15A origin of replication
Modification of the MCS of pSU38delta by inverse PCR with oligo 5'-GCTTCTAGAGTACTAGTAGCGGCCGCTGCAGATTCCGAAGCCCAACCTTTCATA-3' and 5'-AGTACTCTAGAAGCGGCCGCGAATTCTAACATACGAGCCGGAAGCAT-3'
pSB1A3 with an N-terminal Arginine tag. This particular plasmid utilizes the BioFusion (aka Silver Lab) Standard for BioBrick assembly. Adds 10 Arginine residues to the N-terminus of a Protein.
The third vector of the Golden gate series (adapted from psb1c3) This vector expedites golden gate cloning with BBa_K560012, BBa_K560013 and BBa_K560015. To make this set of vector compatible with the standard biobricks, we have maintained PstI and XbaI recognition sites. Flanking these two sites are the cleavage and recognition sites of BsaI, a kind of Type IIS restriction endonuclease. This kind of restriction endonucleases cleaves outside their recognition sites thus creating different sticky ends. If you want to assemble four biobricks at a time, you can put the biobricks in order into corresponding vector by the digestion of PstI and Xbal. Then mix these assembled vectors and a backbone (PFUS4 from TALEN golden gate kit). Under the effect of ligase and Bsal, these biobricks will have sticky ends nose to tails and ligate in order in a single reaction.
Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting, Nucleic Acids Research, 2011, Vol. 39, No. 12.
This is pSEVA321 converted into a biobrick compatible format by substituting the original multiple cloning site for biobrick prefix and suffix. pSEVA321 is a broad host range plasmid, capable of replication in E.coli and several other organisms. It is capable of replication in the cellulose producing bacterium Gluconacetobacter xylinum. We have used it as our standard backbone for G.xylinum genetic engineering.
The original pSEVA331 backbone comes from the SEVA (Standard European Vector Architecture) collection.
Broad host range BioBrick plasmid derived from pRL1383a with low to medium copy number
RSF1010 is a broad-host-range plasmid first described in 1974 (Guerry 1974), with its entire sequence and gene organization subsequently described in 1989 (Scholz 1989). It is a naturally occurring 8.6kb broad-host-range plasmid in the E. coli incompatibility group Q. The conjugative transfer and stable replication of this plasmid are possible due to the mob genes with the associated origin of transfer (oriT) and the rep genes with the associated origin of vegetative replication (oriV), respectively. RSF1010 derived plasmids which include the oriV and associated Rep proteins are stably maintained in Pseudomonas (Bagdasarian 1981), Caulobacter (Umelo-Njaka et al. 2001), Erwinia, and Serratia (Leemans 1987). In addition, RSF1010 derived plasmids including the oriV, oriT and its associated rep and mob genes are transferred by conjugation to at least four cyanobacteria strains (Mermet-Bouvier 1993). These cyanobacteria strains include Synechocystis PCC6803 and PCC6714 and Synechococcus PCC7942 and PCC6301. A number of RSF1010 derived plasmids have been constructed due to the utility of this broad-host-range plasmid. For example, pSB2A, containing a 5.6kb RSF1010 derived region including the necessary mobilization and replication regions, can be transferred through conjugation to and stably maintained in Synechocystis PCC6803, PCC6714, and Synechococcus PCC7942 and PCC6301 (Marraccini 1993). The RSF1010 plasmid used in this study, pRL1383a was constructed for use in genomic studies of the diazotrophic, multicellular cyanobacterium Anabaena PCC 7120. This plasmid contains the mob and rep regions necessary for conjugation and autonomous replication, respectively. Additionally this vector is made resistant to Streptomycin and Spectinomycin due to the presence of the aadA gene (Wolk 2007). This plasmid can be mobilized by the E. coli derived self-transmissible plasmid RP4. Mobilization genes are not necessary for transfer of a mobilizable plasmid if the self-transmissible plasmid and the mobilizable plasmid share a common origin of transfer (Snyder and Champness 2007). The origin of transfer for RP4 is 99 base pairs and contains binding sites for transfer proteins encoded by RP4. For more information and a list of references, please visit: http://2008.igem.org/Team:Hawaii/Project/Part_A#Literature_Cited
The original pRL1383a MCS has been replaced by the BioBrick BBa_B0034 and its flanking BioBrick restriction sites.
pSBBs4S-Pxyl: Integrative expression vector for Bacillus subtilis</i
This part is an expression vector for Bacillus subtilis. It integrates at the thrC locus. It has a ampicillin resistance for cloning in E.coli and chloramphenicol resistance for selection in B.subtilis. The multiple cloning site downstream of a Pxyl promoter, that is inducible with xylose. This BioBrick is part of the LMU 2012 igem project Bacillus BioBrick Box http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR. The clonging site also includes a rfp for selection for cloning in E.coli.
This part is an igem compatible version of the Bacillus expression vector pXT
pcDNA3.1+ puro/ ΔpCMV, based on pcDNA3.1+; no promoter; amp, puro
V0120, standard high-copy cloning vector for E. coli
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).
pSB1c3 is changed to pSB1Pe by replacing the antibiotic gene with proteorhodopsin. The plasmid also has a carbon stress promoter- PcstA and double terminator - B0015. The EXSP sites are placed within the promoter and the double terminator region so as to allow cloning of recombinant protein into the plasmid and expressing the same in low carbon conditions.
The reading frame should be considered while designing
Plasmid Backbone - Psb1c3 Parts - B0015, K118011
Intermediate in the construction of P_template1
pORE GFP Reporter Series Vector with BioBrick MCS (Kan resistance)
Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Kan resistance in plants. Has constitutive plant promoter and in-frame GFP reporter. Conforms to Silver BIoBrick Assembly Standard 21.
Modified vector pORE_R2 from the Arabidopsis Information Resource
VP64-NLS-pcDNA3.1+, pcDNA3.1+ with Eco/Spe NLS BioBrick inserted into Eco/Xba; amp, neo
possible biobrickable terminator with polyA signal terminator
Digestion product of ApaI and HindIII building bloc variation
pSB1A3_Int (BamHI, XhoI, NdeI, NheI added)
This part is based on pSB1A3, with BamHI, XhoI, NdeI and NheI sites added through site-directed mutagenesis. This plasmid backbone retains compatibility with BioBricks (except with RFC 21 - BamBgl), its antibiotic resistance, and its high copy number in E. coli. The additional restriction sites allow the addition of DNA sequences homologous to genomic sequences in Synechocystis sp. PCC 6803 and other species, enabling the plasmid to be used to integrate the BioBrick construct and an antibiotic resistance gene into the chromosome at a specific location through homologous recombination. Integration sequences can be designed so that the BioBrick construct is added in the middle of a gene knocking the gene out in the process, or between two genes without interfering with native expression. The BioBrick construct can be assembled in E. coli cells without changes to the standard procedure, and when completely assemble, the plasmid can be introduced into the target species. Synechocystis sp. PCC 6803 naturally takes up DNA from its environment, and if sequences are homologous to regions of the genome, will naturally undergo homologous recombination. Other species may lack one or both of these abilities, and the addition of another plasmid carrying a recombinase gene may be necessary for genomic integration.
BamHI, XhoI, NdeI, and NheI sites were mutated in using Stratagene's site-directed mutagenesis kit, the sites were mutated in by changing the nucleotide sequence, without the addition or removal of nucleotides.
pSB1A3 with BamHI, XhoI, NdeI, and NheI sites mutated in outside of the terminator regions.
pORE Open Serices Vector with BioBrick MCS (Kan resistance)
Vector for transformation of plants. Cloning in E. coli, transfer to Agrobacterium, integrates into plant genome. Kan resistance marker in bacteria, confers Kan resistance in plants. Has no promoter or reporter. Conforms to Silver BioBrick Assembly Standard 21.
Modified vector pORE_O1 from the Arabidopsis Information Resource
pSB1C3 with RBS-RFP-TT between SpeI-PstI
Nicolo' Politi, Susanna Zucca, Tommaso Goggia, Paolo Magni
When the SpeI-PstI DNA fragment from BBa_J61002 , containing the mRFP1 gene with a strong RBS and a double terminator, is transferred into pSB1C3 (digested SpeI-PstI) the resulting plasmid is BBa_K516999. See pSB1C3 for detailed feature information.
pSB1C3 (backbone) and BBa_J61002 (SpeI-PstI insert).
FRT-marked mam. transfection vector (from Invitrogen pcDNA5 FRT/TO); no promoter; amp, hygro
BioBrick integrative base vector for S. cerevisiae
Susanna Zucca, Lorenzo Pasotti, Federica Sampietro, Paolo Magni
Warning, this plasmid does not have a proper BioBrick cloning site.
"A new plasmid vector, pSCANS, has been constructed which allows rapid generation of an ordered set of nested deletions from either strand of a cloned DNA fragment. pSCANS is based on the low-copy F replicon. The size of the vector DNA has been reduced to the 4.4-kbp range by removing the 2.5-kbp sop (stability of plasmid genes) region. The resulting plasmid has the low copy number typical of F plasmids and it remains stable enough to be easily maintained by growth in the presence of kanamycin, the selective antibiotic. DNA in amounts convenient for sequencing is readily obtained by amplification from an IPTG-inducible P1 lytic replicon. The vector's multiple cloning region (MCR), which has several unique sites for both shotgun and directional cloning, is flanked on one side by recognition sequences for the extremely rare cutting intron encoded nucleases I-CeuI and I-SceI, and on the other side by a recognition sequence for another intron encoded enzyme, PI-PspI and a nicking site for the phage f1 protein, gpII, that initiates f1 rolling circle DNA replication. Cleavage with the intron encoded enzymes leaves four-base 3' overhangs that are resistant to digestion with E. coli ExoIII. Between these sites and the MCR are recognition sites for several rare 8-base cutters that leave ExoIII sensitive termini. Double cutting with one intron encoded enzyme and an adjacent rare cutting restriction endonuclease allows for unidirectional 3' to 5' digestion across the insert with ExoIII. Alternatively, plasmid linearized with I-SceI can be blunt ended to produce an ExoIII sensitive end an ent generates a good distribution of deletion clones following electroporation. Deletion clones are sized and sequenced using vector specific forward and reverse primers. "
pSB4K15 is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and kanamycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
The resistance cassette is terminated by a Lux bidirectional terminator. Adjacent to this terminator there is a designed primer binding site to where the forward primer for lambda red can be designed to bind to no matter what the insert in the cloning site is or what the resistance is. The resistance cassette is flanked by SalI and SacI restriction sites for easy switching of resistance in the backbone. The pSC101 origin of replication is flaked by NheI and MluI restriction sites for easy switching of origin of replication.
Biobricked pCS2+ plasmid with Elastase promoter for frogs and chicken
This plasmid is a biobricked version of the plasmid pSC2+(K812000)with Elastase promoter. This plasmid is made of a bacterial ampicilin resistant backbone. The Biobrick site is surrounded by a 5'UTR expression enhancer, and a poly-A tail with a SV40 3' UTR that enhance the stabilisation of the mRNA and the expression of the protein. This plasmid is meant to be micro-injected in frog eggs or chicken eggs. This plasmid does not replicate, so it gets diluted and degraded as the animal grow, leaving no chance for DNA propagation to the breeding. To use this plasmid, you simply have to insert your protein between the EcoRI and PstI site. It is better to add a kozac sequence in front of your gene to enhance its expression. This plasmid contains the standard registry insert J04450 as a negative cloning control.
CMV of pCS2+ (K812000) was replaced by Elastase using the double digestion SalI and HindIII. Then J04450 was replaced by sfGFP using the Biobrick K812031 with the double digestion EcoRI and PstI.
The plasmid template comes from K812000 and the constitutive CMV promoter was replaced by the Elastase promoter. The sfGFP comes from K812031. sfGFP comes from : Love, N.R. et al., 2011. pTransgenesis: a cross-species, modular transgenesis resource. Development (Cambridge, England), 138(24), pp.5451???8
NLS-pcDNA3.1+, pcDNA3.1+ with Eco/Spe NLS BioBrick inserted into Eco/Xba; amp, neo
pSB4A15 is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and ampicillin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
The plasmid has a suicide gene and a mutation-controller sequence. It can hence eliminate devious mutations and help to restrain biobricks to only our engineered organisms.
Do not use commonly used commercial competent cells when using this plasmid. Special strains are required.
Contains the mRFP cassette (J04450) and additionally terminators and flanking region from pSB1C3. The origin of replication has been replaced with CloDF13 replicon with a copy number of 20 ??? 40 per cell. The cat gene has been replaced with aadA conveying spectinomycin and streptomycin resistance.
The terminators in the pSB1C3 made gibson assembly difficult and as such we switched to traditional cloning using XhoI and AvrII cut sites.
Upgraded pSB1C3 swapping out the origin and resistance cassettes for CloDF13 and aadA from pCDF
pSB1AT3 is a high copy number plasmid carrying ampicillin and tetracycline resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell).
pSBbs1AK1 (replicative Bacillus subtilis expression vector)
Max Schelski, Niklas Schmacke, Cathleen Hagemann, Sophia Mädler
This part is an expression vector for Bacillus subtilis. It is replicative both in E.coli and B.subtilis. It has an kanamycin resistance for cloning in E.coli and ampicillin resistance for selection in B. subtilis.
A double resistance for selection in both B.subtilis and E.coli was needed
This part is similar to [http://partsregistry.org/Part:BBa_J61002 J61002] but the backbone is based on pSB1C3 and carries a chloramphenicol resistance.
* This part is a compination of E1010, B0034 and pSB1C3 from the registry.
BioBrick integrative base vector for E. coli
Lorenzo Pasotti, Matteo Meroso, Nicolo' Politi
pSB4C15 is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and chloramphenicol antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
pSB3C5 + After TALEN (K1480001) Target site
Backbone vector of pSB3C5 that contains a single modification with a TALEN target site after the origin of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains a single TALEN target site (AF).
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)
pACYC177 converted into BioBrick vector (formerly BBa_I739202)
partial ampicillin resistance marker (nonfunctional)
We use LacZ to report the work of gene circuit.
LacZ is from S.cerevisivae. pYE is bought from invitrogen.
This plasmid backbone is based on pMA from Geneart. It has BioBrick prefix and suffix flanked on both sides by 50 bp of homology arms for insertion of the contained BioBrick into the 00500 locus of pCC2FosMB (BBa_K260001) by recombineering. The pMA-@CC2FosMB-00500bp backbone is available with BBa_K260015, which is composed of promoter-FRT-dhfr. If the PvuII fragment from BBa_K260004-BBa_K260015 is transferred by recombineering to the the 00500 locus of BBa_K260001-BBa_K260016, the intervening sequence between the FRT sites of BBa_K260015 and BBa_K260016 on the BBa_K260001 backbone will be 500 bp exactly. These plasmid backbones are similar and all allow recombineering-mediated transfer of the contained BioBrick (typically BBa_K260015) to the pCC2FosMB backbone BBa_K2600001. They differ in the homology arms, which determines the locus of insertion into pCC2FosMB. The bp numbers denote final inter-FRT distance as described above: BBa_K260004: pMA-CC2FosMB-00500bp BBa_K260005: pMA-CC2FosMB-01000bp BBa_K260006: pMA-CC2FosMB-02000bp BBa_K260007: pMA-CC2FosMB-05000bp BBa_K260008: pMA-RQ-CC2FosMB-10000bp
The 50 bp homologies were designed to delete 100 bp of BBa_K260001-BBa_K260016 and put the FRT site of BBa_K260015 (if used with the present backbone BBa_K260004) at a defined distance from the FRT site of BBa_K260016.
BBa_K260001 (pCC2FosMB) 3' homology FRT-00500bp
BBa_K260001 (pCC2FosMB) 5' homology FRT-00500bp
pSBLb4E15 E. coli and lactobacilli shuttle vector with erythromycin resistance
E. coli Lactobacilli shuttle vector pSBLb4C15 is a shuttle vector containing the ori pSH71 originating from the plasmid pJP059 which was taken from Lactococcus lactis. It has a red insert.
Had to make sure it was compatible with biobrick standard 10
Ori in pEL4C15 replaced with ori from pJP059. Chloramphenicol resistance replaced with erythromycin resistance.
Eukaryotic terminator vector (formerly BBa_J52017)
This vector is made of BioBrick vector pSB1AK3, which has eucaryotic terminator added.
pSB1A2 is a high copy number plasmid carrying ampicillin resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). pSB1A2 has one terminator upstream of its MCS, which is oriented to prevent transcription from *inside* the MCS from reading out into any DNA outside the MCS. The orientation of the pBLA promoter driving the AmpR gene is such that relatively little transcription should be coming into the MCS from upstream. Ideally, a future version of the standard biobrick vectors would have terminators bracketing an MCS that were 100% efficient in terminating both into and out of the MCS region.
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.
This part was created from BBa_J61035 when it was cut with EcoRI and SpeI and this section inserted before a terminator BBa_B0015 on a pSB1AK3 plasmid. The part was never created in the pure form since when created it carried our part of interest between Xbal and SpeI. Creating it allows multiple parts to be shipped as a parts in this plasmid without further complicating explanations. This should perform a usefull service since many expression parts require the addition of RBS and terminator which is conveniently done using this sequence of events due to the multiple antibiotic resistances collected during the process.
Part from kit provided BBa_J61035 and BBa_B0015.
Verfication forward (VF2) primer binding site
Bacillus shuttle vector used for assembling our fluorescent ratiometric construct. Sequence provided here as reference only.
Bacillus shuttle vector. Includes pSPANK and b0010 terminator, both of which are integrated into our final construct.
Designed and assembled by a member of our supporting lab, PJ steiner.
pSEVA331-Bb - pSEVA331 with biobrick prefix and suffix
This is pSEVA331 converted into a biobrick compatible format by substituting the original multiple cloning site for biobrick prefix and suffix. pSEVA331 is a broad host range plasmid, capable of replication in E.coli and several other organisms. It is capable of replication in the cellulose producing bacterium Gluconacetobacter xylinum. We have used it as our standard backbone for G.xylinum genetic engineering.
Part BBa_K238024, the lock, was sent to us by Geneart in this plasmid. It differs from pMA-RQ in that it has an extra suffix and 4 extra basepares after the second suffix, being TACT, making it 25bp longer.
This plasmid is based on pSC101 and contains the rhamnose operon (rhaR and rhaS genes) as well as the F70L variant of FLP recombinase under rhamnose-promoter control. Therefore L-rhamnose can be used to induce FLP(F70L) expression. The pSC101 origin of replication is temperature sensitive due to a mutation of repA. Permisive temperature is 30 degC. 37 degC should be avoided as the plasmid can not replicate.
This plasmid comes from Sarov M, Schneider S, Pozniakovski A, Roguev A, Ernst S, Zhang Y, Hyman AA, Stewart AF. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nat Methods. 2006 Oct;3(10):839-44. However, it was modified by deletion of dhfr gene and RedABGRecA operon through insertion of a kanamycin resistance cassette.
dhfr had to be deleted as the same gene for trimpethoprim resistance was going to be used elsewhere in the same cell.
Subtilin inducible expression plasmid for Bacillus subtilis
Maarten van den Nieuwenhof, Ramon Sieber, Arend Jan Suk
Brick sequencing primer (3CF) binding site
Brick sequencing primer (3BR) binding site
pGADT7 is the AD Vector included with MATCHMAKER Two-Hybrid System 3.
The pGADT7 vector expresses proteins fused to amino acids 768???881 of the GAL4 activation domain (AD). In yeast, fusion proteins are expressed at high levels from the constitutive ADH1 promoter (PADH1); transcription is terminated at the ADH1 transcription termination signal (TADH1). The fusion protein is targeted to the yeast nucleus by the SV40 nuclear localization sequences that have been added to the activation domain sequence (1). pGADT7 also contains the T7 promoter, an HA epitope tag, and a MCS. pGADT7 replicates autonomously in both E. coli and S. cerevisiae from the pUC and 2 m ori, respectively. The vector carries Ampr for selection in E. coli and the LEU2 nutritional marker for selection in yeast.
Propagation in E. coli: ??? Suitable host strains: DH5a, DH10 & other general purpose strains ??? Selectable marker: plasmid confers resistance to ampicillin (100 mg/ml) to E. coli hosts ??? E. coli replication origin: pUC ??? Copy number: ~500 ??? Plasmid incompatibility group: pMB1/Col E1 Propagation in S. cerevisiae: ??? Suitable host strains: Y187(a), Y190(a), SFY526(a), CG1945(a), HF7c(a), or AH109(a) ??? Selectable marker: LEU2 ??? S. cerevisiae origin: 2 m
Plasmid Backbone including M-13 primer region
This plasmid was ssent from a company that assists iGEM.
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.
kanamycin + ampicillin resistant p15A vector for 2ab assembly
kanamycin + ampicillin resistant p15A vector, used for 2ab assembly referred to internally as pBjh1601KA.
Complete, arabinose inducible phagemid, CmR, p15a vector
Vector contains all of the cis elements required to be an efficient phagemid. Arabinose induces lysis.
Biobricked pSC2+ plasmid with pElastase promoter for frog use
Biobricked pSC2+ plasmid with a pElastase promoter for frogs This plasmid is a biobricked version of the standard plasmid pSC2+ with the pElastase promoter. This is a daugter par of K812001. This plasmid is made of a bacterial ampicilin reststant backbone, with a universal CMV promoter. The Biobrick site is surrounded by a a 5'UTR expression enhancer, and a ploy-A tail with a SV40 3' UTR that tnhance the stabilisation of the mRNA and the expression of the protein. This plasmid is ment to be micro-injected in frog eggs or chicken eggs for a transcient expression of the protein at the early ages of development of the embryo. This plasmid does not replicate, so it gets diluted and degraded as the animal grow, leaving no chance for DNA propagation to the breeding. To use this plasmid, you simply have to insert your protein between the EcoRI and PstI site. It is better to add a kozac sequence in front of your gene to enhance its expression. This plasmid contains the standard registry insert J04450 as a negative cloning control.
This is the daughter part of K812000 where the CMV promoter has been replaced by the pElastase promoter cutting using HindIII and SalI unique site
This plasmid is the biobricked version of pSC2+ where the promoter has been replaced. Check the original part K812001 for more information.
This is a plasmid of pBSK II. Resistant to ampiciline
This part comes from a Genomic Sequence from the NCBI.
Empty plasmid for gene expression in S. cerevisiae. Ura selection in yeast and ampicillan resistance in E. coli. One of a series of pBluescript-based centromere vectors (ATCC 77142 ATCC 77145, ATCC 77157 to ATCC 77158) differing in the yeast selectable marker gene. The order of the major features in this plasmid is: TRP1 - f1 ori (NaeI) - T7 promoter - lacZ'/MCS - T3 promoter - pMB1 ori - bla - CEN6 - ARSH4. Contains promoters for in vitro RNA synthesis, priming sites useful for sequencing, and encodes the lacZ alpha (lacZ') peptide. Restriction digests of the clone give the following sizes (kb): EcoRI--4.8; BamHI--4.8; PvuII--4.2, 0.5. (ATCC staff) Medium is 1227 LB plus ampicillin. NCBI gi: 416309 Hosts: E.coli HB101, Saccharomyces cerevisiae, E.coli, Saccharomyces cerevisiae YPH499, Saccharomyces cerevisiae YPH500, Saccharomyces cerevisiae YPH501. Related vectors: pRS313, pRS315, pRS316.
Eliminated PstI, XbaI and SpeI restriction sites.
From ATCC. Pubmed accession number U03442.
Integrative plasmid for Bacillus subtilis (pSBbs4E)
Vector containing Hisx6 and Sortase tag LPETGG
This vector is constructed to simplify the usage of the transpeptidase Sortase A for future iGEM teams. Following a BamHI restriction site we constructed a synthetic sequence consisting of three main elements: 1) A linker (GSSS) to allow for limited mobility of the attached peptides and facilitate correct folding 2) A Sortase A tag (LPETGG) that is used to conjugate the proteins to poly-glycines 3) A standard Hisx6 tag commonly used for Immobilized Metal Affinity Chromatography (IMAC) This sequence follows a BamHI restriction site which can be used by teams to clone in whichever peptide they would like to conjugate using Sortase A.
BamHI is a very convenient restriction enzyme for our application as its restriction site will code for a glycine and a serine, both of which are often used in linkers and tend to be unproblematic in such sequences.
The plasmid was constructed using the iGEM-supplied pSB1C3 and a synthetically produced sequence.
This plasmid can be used to eliminate an upstream PstI restriction site and a downstream BamHI restriction site in a potential BioBrick part.
It was obtained by performing a PCR reaction amplifying the pSB1C3 plasmid and using appropriate overhangs containing an NsiI site to combine with the PstI site and a BglII site to combine with the BamHI site.
Kanamycin resistant BAC with intact R6K origin (BBa format). Can be maintained at high copy in strains expressing pir Referred to internally as pBacO.
This is a new backbone modified from psb1c3 to facilitate ligation independent cloning. This new backbone allows creation of parts and composites into any desired cloning standard.
The EcoRI and PstI restriction sites are inactivated by mutagenesis so as not to create two sets of these restriction sites. A BsiWI restriction site is created between the XbaI and SpeI restriction sites for ligation independent cloning.
verification reverse(VR) primer binding site
pRS316 plasmid for S. cerevisiae in BioBrick format
Modified pRS316 plasmid for S. cerevisiae. Has selection for tryptophan negative, Ampicillin, and LacZ. Can be used to insert BioBricks into S. cerevisiae.
Used mutagenesis to remove EcoRI site at 389???394. Also used mutagenesis to rearrange the EcoRI, XbaI, SpeI, and PstI into BioBrick format. Added the tryptophan negative gene for selection.
Comes from the genomic sequence of pRS316
pSB1A2 have a double mutation in the 80th serine (TCC->TAG) of the APr gene. This plasmid need a tRNA amber suppressor to express this gene. So in case of horizontal gene transfer this gene won't be expressed and then the plasmid will be lost more easily.
2 oligo mutated on 2 positions were used for this purpose.
This gene is mutated by the PCR quick change mutagenesis.
Expression Plasmid coding for His-tag, also containing a thrombine cleavage site between coding region and Histag, so that His-tag can be cleaved off. Vecotr opening with PstI/XmaI, clone part into it with PstI/NgoMIV. Ampicillin resistance.
very strong RBS 6xHis-Tag Thromin-cleavage-site NgoMIV-compatible restriction site for in-frame insertion
pSB1A3 with an C-terminal Arginine tag. This particular plasmid utilizes the BioFusion (aka Silver Lab) Standard for BioBrick assembly. Adds 10 Arginine residues to the C-terminus of a Protein (Includes Stop Codon)
The second vector of the Golden gate series (adapted from psb1c3) This vector expedites golden gate cloning with BBa_K560012, BBa_K560014 and BBa_K560015. To make this set of vector compatible with the standard biobricks, we have maintained PstI and XbaI recognition sites. Flanking these two sites are the cleavage and recognition sites of BsaI, a kind of Type IIS restriction endonuclease. This kind of restriction endonucleases cleaves outside their recognition sites thus creating different sticky ends. If you want to assemble four biobricks at a time, you can put the biobricks in order into corresponding vector by the digestion of PstI and Xbal. Then mix these assembled vectors and a backbone (PFUS4 from TALEN golden gate kit). Under the effect of ligase and Bsal, these biobricks will have sticky ends nose to tails and ligate in order in a single reaction.
Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting>>Nucleic Acids Research, 2011, Vol. 39, No. 12
Variant of pBca1021 (J61003) with GFP inserted. Part J23019 is the same as J23018 but in pSB3C6.
chloramphenicol acetyl transferase (CmR resistance marker)
This plasmid backbone is the same as BBa_K245004 except that it doesn't contain His tag at all.
First, vector BB-NIC-II-HisN was cut with EcoRI and XbaI and insert with appropriate part of prefix modified according to standard 37 without His tag was ligated in between.
The basis for BB-NIC-II-NC is vector pSB1A2. Modified prefix and suffix are of synthetic origin. ccdB originates from part BBa_P1010.
pSB1C5 (1bp mutation of pSB1C3 to remove SacI)
This part is a plasmid bone, which has only a single site mutation compared with pSB1C3. The SacI site is mutated to allow this plasmid compatible to BBF EFC 53 standard.
In order to make our working plasmid compatible to BBF RFC 53, we search the restriction enzymen site in pSB1C3 plasmid. And we found a SacI site in the pSB1C3. The site is between downstream of chloramphenicol and the T0 terminator. We think the mutation does not affect the regular function of pSB1C3 plasmid. So all of our following experiment of fusion protein was done in this new plasmid.
this part comes from the partsregistry BBa_pSB1C3
pcDNA3.1+ hygro/ΔpCMV, based on pcDNA3.1+; no promoter; amp, hygro
vector pYE, designed for inducible expression of recombinant proteins in S.cerevisivae.
pYES2 is a 5.9 kb vector for inducible expression of recombinant proteins in S.cerevisivae. Feature of the vector allow easy cloning of your gene of interest and selection of transformants by uracil prototrophy. The vector contains the follow elements:
We use this vector for expressing mutant Tor2. In order to operate easily, we separate Tor2 into three parts. So we reconstruct the pYE by adding specific restriction sites for linking.
Golden brick version of pSB1C3 plasmid for Golden Gate and Golden Brick cloning
This is a modified version of pSB1C3 from the registry with the J4450 negative control. The biobrick sites are modified to incorporate a BsaI restriction site that enable to the use of golden gate cloning and golden brick cloning to assemble full contructs in a single shot of digestion/ligation. The complete protocol of the goldengate can be find in the original paper: Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes. PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 The Golden Brick assembly is an invention of the iGEM Evry 2012 Team. A complete description of the procedure can be find on our website: http://2012.igem.org/Team:Evry/GB
The negative control J4450 has been amplified with new goldenbrick extensions using PCR and re-inserted in pSB1C3 using EcoRI and PstI cloning.
This part is a derivation of the pSB1C3 plasmid from the registry.
pSB3C5 + MID TALEN (K1480001) Target site
Backbone vector of pSB3C5 that contains a modification with a TALEN target site on the center of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains a single TALEN target site (MID).
Toolbox Institute of Biology II / BIOSS Center for Biological Signalling Studies Albert-Ludwigs-Universit??t Freiburg Germany
pGA3K3, Gibson assembly plasmid (bglBrick) with pLac-GFP insert
pUC57 backbone - digested between BamHI and XbaI. (reverse-complement sequence)
Truncated pSB1C3 with no Prefix or Suffix
This part is a truncated version of pSB1C3 which does not contain a prefix or suffix. It is made to be used as a construction intermediate for the creation of new plasmid backbones which want to insert additional regions at either end of the backbone before replacing the prefix and suffix at either end of the backbone. It is used in K1795023.
We removed the prefix and suffix from pSB1C3 so we could add additional regions on either end of the backbone before replacing the prefix and suffix.
Biobricked pCS2+ plasmid for frogs and chicken
This plasmid is a biobricked version of the standard plasmid pSC2+. This plasmid is made of a bacterial ampicilin reststant backbone, with a universal CMV promoter. The Biobrick site is surrounded by a a 5'UTR expression enhancer, and a ploy-A tail with a SV40 3' UTR that tnhance the stabilisation of the mRNA and the expression of the protein. This plasmid is ment to be micro-injected in frog eggs or chicken eggs for a transcient expression of the protein at the early ages of development of the embryo. This plasmid does not replicate, so it gets diluted and degraded as the animal grow, leaving no chance for DNA propagation to the breeding. To use this plasmid, you simply have to insert your protein between the EcoRI and PstI site. It is better to add a kozac sequence in front of your gene to enhance its expression. This plasmid contains the standard registry insert J04450 as a negative cloning control.
The biobrick site has been inserted in order to obtimize the expression of the protein.
This part is the biobricked version of a standard plasmid (see http://sitemaker.umich.edu/dlturner.vectors/home). We obtained this version from Nicolas Pollet lab and it has been biobricked by our team.
Plasmid pSB1A2 with BsaI site removed from Ampicillin resistance gene. For use in Golden Gate Assembly with BsaI.
Original amino acid sequence of Amplicillin gene retained.
pSB8S15 is a BioBrick standard vector with low copy pSC101ts replication origin (~5 copies per cell) and spectinomycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 repelication origin and propagates at 30???C but stop replicating at 42???C. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
pUC19 with a BioBrick cloning site (Replaced by pSB1A3)
This plasmid is pUC19 with a BioBricks cloning site (EcoRI-NotI-XbaI-SpeI-NotI-PstI) located between the EcoRI and BamHI sites in the multiple cloning site of pUC19.
It is not entirely clear whether there are two contigous copies of EcoRI restriction site or one copy of the EcoRI in this vector. Sequence information did not include this section of the vector. In this part, only a single EcoRI restriction site is shown.
pUC19 is an extremely common cloning vector. This particular version is entered in the registry because several parts synthesized by Codon Devices (BBa_P1002, BBa_P1003, BBa_P1004, BBa_P1005, BBa_I50040) were returned in this vector. Sequence information can be found at http://www.fermentas.com/techinfo/nucleicacids/sequences/puc19.txt
pSB1A3_IntK - General Integration Plasmid with KmR
High copy BioBricked M13 phagemid vector
This biobrick backbone has the high copy number pUC19 origin of replication and an Ampicillin resistance marker. Part also contains the M13 origin of replication which allows the phagemid to be taken up by the M13 phage. Vector does not contain any expressible phage genes; therefore, a Helper Phagemid is required for phage production. Vector contains the following: iGEM VF2 and VR sequencing sites T7 promoters that flank the MCS Translational stop sequences (BBa_B0042) that flank the MCS Transcriptional terminator BBa_B0055 upstream of the MCS Transcriptional terminator BBa_B0054 downstream of the MCS
Plasmid was designed to have the iGEM VF2 and VR sequencing sites along with translational and transcriptional terminators flanking the MCS.
Limtus28i phagemid vector from NEB. Areas flanking MCS were cloned from pSB3C5 backbone.
pSB1AC3-HySp: Integrational backbone into B. subtilis amyE locus with IPTG inducible promoter
We used the amyE intergrational backbone form Munich 2012 (BBa_K823023) that had no promoter. We've added the Hy_Spank IPTG inducable promoter from Rudner's Lab 2004. The Hy_Spank promoter is right in front of the prefix so any biobrick can be inserted in this backbone.
In pDR111 the Hy_spank promoter and the lacI gene are orientated in the same direction. In order to have the Hy_spank promoter right in front of the prefix we turend the promoter around.
iGEM Munich 2012 (Rudner Lab 2004) and Rudner's Lab 2004 (pDR111)
This fosmid backbone was generated by recombineering-mediated transfer of the HincII fragment from BBa_K260016 (inside backbone BBa_K260010) to pCC2FosM (BBa_K260000). This reaction also transferred the blasticidin resistance gene (BsdR), which is now part of this backbone. pCC2FosMB is available with part BBa_K260016 between prefix and suffix.
The insertion site for BsdR was chosen to be upstream of MECP2 exon 1 but still with more than 10 kb of MECP genomic sequence upstream.
Based on BBa_K260000, so it also contains human MECP2 genomic sequence, but has an insert taken from BBa_K260010.
pSB1A11, reconstructed from pSB1A2 with NcoI, XhoI, BamHI and PstI
pSB1A2 is a existing BioBrick, and we reconstruct it to make our connection easier. We add NcoI, XhoI, BamHI, and PstI into the vector.
In order to make our mutation work easier, we separate Tor2 into three parts. so we add fout certain restriction sites into the vector to make the connection easier to conduct.
pSBBs1C-lacZ (lacZ reporter vector for B. subtilis)
This part is a backbone vector for Bacillus subtilis. It integrates at the amyE locus. It has a Ampicillin resistance for cloning in E.coli and Chloramphenicol resistance for B.subtilis. The multiple cloning site is followed by a lacZ gene for reporter assays. This BioBrick is part of the LMU 2012 igem project Bacillus BioBrick Box http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
This reporter vector can be used with the normal BioBrick assembly strategies.
This vector is the Bacillus vector pAC6 brought into the iGEM BioBrick standard.
Biobricked plasmid backbone pRS314 with XbaI, PstI and SpeI sites removed from outside the MCS. One of a series of pBluescript based yeast centrometer vectors developed by Sikorski R.S., Hieter P (Genetics 122:19-27(1989)). Confers Trp resistence in yeast and ampicillan resistance in E. coli where it can be transformed and amplified for cloning purposes.
Removal of non-biobrick compatible restriction sites.
Unmodified version can be found by accession number U03440.
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.
ready-to-inject backbone for T3SS, SlrP taged, with Bsa I cloning site
ready-to-inject backbone for T3SS. Used for protein assembly and expression.
Removed Spe1 restriction site from GSK tag by silent mutation.
SlrP: S. enterica Typhimurium GSK: Glycogen Synthase Kinase 3beta Julie Torruellas Garcia, Franco Ferracci, Michael W. Jackson,1 Sabrina S. Joseph, Isabelle Pattis, Lisa R. W. Plano, Wolfgang Fischer, and Gregory V. Plano. 2006. Measurement of Effector Protein Injection by Type III and Type IV Secretion Systems by Using a 13-Residue Phosphorylatable Glycogen Synthase Kinase Tag. Infect Immun.Vol.74:5645-57.
pYES2 - galactose-inducible expression vector for yeast
S. cerevisae test vector for actuators. Has a Gal1 promoter which is galactose inducible and glucose repressible. Comes with high copy number origin, kanamycin resistance, and leucine yeast selection; can replace with low copy number origin if ordering from DNA 2.0.
Has S. cerevisiae specific LEU selection and 2 um origin of replication allowing for 50-100 copies of the plasmid in yeast cells.
Vector is based off of pJ1201-03c plasmid developed by DNA2.0.
This plasmid is based on pProTetE333 (clontech). A SpeI site was removed via an A-T mutation at position 1166 on the original plasmid, resulting in pProTetE333b. The G00100-G00101 fragment of pSB4A3-1 which includes P1010 between a biobricks multiple cloning site which itself is flanked by + strand transcriptional terminators was then cloned between the XhoI and AvrII sites of pProTetE333b. The resulting plasmid carries a ColE1 origin of replication, a Chloramphenicol resistance marker, the biobricks MCS and the ccdAB death cassette.
pProTetE333 (clontech) and biobricks parts
pGA1A3, Gibson assembly plasmid (bglBrick) with pLac-GFP insert
Non-standard backbone for luciferase construct
This is the sequence of the non-standard backbone that our luciferase construct, BBa_k911004, was synthesised in. It could not be assembled in the high copy number standard psb1c3 backbone due to toxicity issues. Attempts to assemble the 9kb insert into psb3c5, a low copy number biobrick plasmid have so far been unsuccessful. The sequence of the low copy number custom backbone is provided here as a reference.
We did not design this sequence - however, it was chosen to assemble in as it is maintained at a low copy number, avoiding fatal toxicity.
Plasmid for assembly of open reading frame basic parts. A Tet promoter (r0040) and strong ribosome binding site (b0034) lie between the EcoRI and XbaI sites. Insertion of an open reading frame basic part into the XbaI and PstI sites results in constitutive expression but retains basic part utilityin standard assembly.
PCR KB001/ca1021R on pSB1A2-J01022 (250 bp, EcoRI/XbaI) sub into pSB1A2-E0040(GFP;DNA-1-5H) (EcoRI/XbaI) Product is pBca1021-E0040 ---- ca1021R Reverse b0034 XbaI AGCCATCTAGAATTTCTCCTCTTTCTCTAG KB001 Forward EcoRI to construct Ptet part-making biobrick plasmid ttctggaattcgcggccgcaactagagccaggcatc
pSB1C3 + MID, AF and BF TALEN (K1480001) Target sites
Backbone vector of pSB1C3 tha contains a modification with a TALEN target site on the center, before and after the origin of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains three TALEN target sites (MID+AF+BF)
Modifications were inserted by PCR reactions in both strands.
BB-NIC-II-HisN originates from BioBrick vector pSB1A2 but contains modified prefix and suffix according to assembly standard 37. T7 promoter, RBS and ATG are included in prefix and T7 terminator and STOP codon in suffix. His tag is also inserted between ATG and multiple cloning site in prefix. There are four new inner restriction sites added in multiple-cloning site (NgoMIV and AgeI in prefix; XmaI and BspEI in suffix) which enable extension of part cloned into BB-NIC-II-HisN with SG on N-terminal or TG on C-terminal; extension of linker and creating a friendly scar during parts fusion.
BioBrick vector pSB1A2 was first cut with EcoRI/PstI. An insert containing modified prefix and suffix (according to assembly standard 37) was ligated in between. Subsequently a ccdB domain was inserted between AgeI and XmaI restriction sites.
The source is BioBrick vector pSB1A2; modified prefix and suffix sequences are of synthetic origin; ccdB originates from part BBa_P1010.
The low copy backbone, pSB3K3 has a BsaI site at position 2498. Through site-directed mutagenesis, we altered the base pair 2498 G>A, so that it would be Golden Gate assembly-compatible.
The sequence immediately downstream of the p15a origin in this plasmid is an almost exact match to the AmpR cassette (see BBa_P1002). It seems likely that the orignal definition of the p15a origin was generous and could likely be truncated.
The original pSB3K3 backbone was derived from the parts registry.
pSB3C5 + Before TALEN (K1480001) Target site
A biobrick shuttle plasmid working between Lactobacillus plantarum, and E.coli with erythromycin res
It's a biobrick plasmid with biobrick assembly restriction sites, a fused origin of replication between gram positive and negative cells, and an erythromycin resistance.
A part of pSIP409 was extracted with tails and re-circularized.
pSBBs3C-luxABCDE (lux reporter vector for B.subtilis)
This part is a reporter vector for Bacillus subtilis. It integrates at the sacA locus of B.subtilis genome. It has a ampicillin resistance for cloning in E.coli and Chloramphenicol resistance for selection in B. subtilis. The multiple cloning site has a rfp for selection and is followed by the luxABCDE gene cluster for luminescence reporter assays. This BioBrick is part of the LMU-Munich 2012 igem project Bacillus BioBrickBOX http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
This vector is an igem compatible version of the pAH328 Bacillus reporter vecotr.
pcDNA3.1+ zeo/ ΔpCMV, based on pcDNA3.1+; no promoter; amp, zeo
pSB1K30: High copy BioBrick cloning/expression backbone carrying Kan resitance
pSB3C5 is a low to medium copy plasmid based with chloramphenicol resistance. It has a p15A-derived replication origin. pSB3C5 is derived from the BioBricks vector scaffold BBa_I51001.
BBa_I51001 BioBricks vector scaffold, p15A based origin and a version of the chloramphenicol resistance cassette that has been cleaned of restriction sites.
pSB1A2 w/ "T" at -32 upstream from RNAII
This is a modified version of plasmid pSB1A2 (http://parts.igem.org/Part:pSB1A2). The position 32 bases upstream from the beginning of the RNAII transcript has been changed from a "C" to an "T". Mutations at this position have been shown to affect plasmid copy number in Castagnoli, 1985 (http://www.ncbi.nlm.nih.gov/pubmed/3895161).
Part was created by ligating pSB1A2 iPCR product to annealed oligos with an "N" at the -32 position.
pBs4S: Bacillus subtilis vector, thrC integration, spec resistance
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the thrC locus and can be selected with Spectinomycin cassette. It has a ampicillin resistance for cloning in E.coli. This backbone is a BioBricked version of the B. subtilis vector pDG1731.
This vector is compatible with pBS1C and pBS2E in one strain.
This BioBrick is part of the LMU 2012 igem project BacillusBioBrickBox For handling of B. subtilis vectors, please see here. The transformation into B. subtilis is explained here.
The plasmid pGAPzalphaC was standardized according to iGEM standards using a linker piece
This is the common plasmid backbone for all the miRNA sensors used in our project. They contain both ampicillin and kanamycin resistance marker.
pBBknock standard vector for allelic replacement
Michela Casanova, Lorenzo Pasotti, Susanna Zucca, Nicolo Politi, Ilaria Massaiu, Cinzia Calvio, Gabriella Cusella, Paolo Magni
This vector can be specialized to target the desired locus in the Escherichia coli genome and carry out allelic replacement, e.g., knockout, chromosomal integration, mutagenesis, etc. See Casanova et al. (2016), Biol Proced Online for detailed instructions and protocols.
Synthetic DNA obtained via GenScript synthesis service.
This improved T7 promoter has not only promoter region but also Histigine Tag sequence. This promoter let you correct and purify proteins by using His Tag antibody
This parts is synthesized artificially.
pSB3C5 + AF and BF TALEN (K1480001) Target sites
Backbone vector of pSB3C5 that contains two modifications with a TALEN target site after and before the origin of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains two TALEN target sites (AF+BF)
ampicillin resistant split R6K vector. Requires expression of pir to replicate. Referred to internally as pBjk2993.
ampicillin resistant p15A vector referred to internally as pBjo1601A
pSB1A2 w/ "G" at -32 upstream from RNAII
This is a modified version of plasmid pSB1A2 (http://parts.igem.org/Part:pSB1A2). The position 32 bases upstream from the beginning of the RNAII transcript had been changed from a "C" to an "A". Mutations at this position have been shown to affect plasmid copy number in Castagnoli, 1985 (http://www.ncbi.nlm.nih.gov/pubmed/3895161).
We used the backbone constructed our plasmid in
Mammalian test vector for sensors. Has a Comet protein coding sequence which produces a GFP-like protein. Comes with high copy number origin and ampicillin resistance; can replace with low copy number origin and kanamycin resistance if ordering from DNA 2.0.
All of the parts were designed by BIOE44 instructors in conjunction with DNA 2.0.
pcDNA3.1+ neo/ ΔpCMV, based on pcDNA3.1+; no promoter; amp, neo
DESCRIPTION: This plasmid backbone is based on pMA from Geneart. It has BioBrick prefix and suffix flanked on both sides by 70 bp of homology arms and a blasticidin resistance cassette (BsdR) for insertion of the contained BioBrick into the proper locus of pCC2FosM (BBa_K260000) by recombineering using the HincII fragment including the BioBrick. The product of this reaction is the pCC2FosMB backbone (BBa_K260001), which then contains the same BioBrick that was inside the present backbone BBa_K260010 before, with BioBrick prefix and sufix maintained intact. Initially this was done with the TT_FRT_GFP BioBrick (BBa_K260016) using BBa_K260010-BBa_K260016. The pMA-@CC2FosMB-00500bp backbone is available with BBa_K260015, which is composed of promoter-FRT-dhfr. If the PvuII fragment from BBa_K260004-BBa_K260015 is transferred by recombineering to the the 00500 locus of BBa_K260001-BBa_K260016, the intervening sequence between the FRT sites of BBa_K260015 and BBa_K260016 on the BBa_K260001 backbone will be 500 bp exactly. These plasmid backbones are similar and all allow recombineering-mediated transfer of the contained BioBrick (typically BBa_K260015) to the pCC2FosMB backbone BBa_K2600001. They differ in the homology arms, which determines the locus of insertion into pCC2FosMB. The bp numbers denote final inter-FRT distance as described above: BBa_K260004: pMA-CC2FosMB-00500bp BBa_K260005: pMA-CC2FosMB-01000bp BBa_K260006: pMA-CC2FosMB-02000bp BBa_K260007: pMA-CC2FosMB-05000bp BBa_K260008: pMA-RQ-CC2FosMB-10000bp SOURCE: Geneart plasmid pMA, with 2x 70 bp from pCC2FosMB K260001. DESIGN: The 70 bp homologies were designed to delete 100 bp of BBa_K260001-BBa_K260016 and put the FRT site of BBa_K260015 (if used with the present backbone BBa_K260004) at a defined distance from the FRT site of BBa_K260016.
The 2x 70 bp homologies were designed to delete 100 bp of pCC2FosM (Part:BBa_K260000). The insertion site on pCC2FosM was chosen to be upstream of MECP2 exon 1 but still with more than 10 kb of MECP genomic sequence upstream.
Geneart plasmid pMA, with 2x 70 bp from pCC2FosM (BBa_K260000).
BB-NIC-II-HisN originates from BioBrick vector pSB1A2 but contains modified prefix and suffix according to assembly standard 37. T7 promoter is included in prefix and T7 terminator in suffix. There are four new inner restriction sites added in multiple-cloning site (NgoMIV, AgeI-prefix; XmaI/BspEI-suffix) which enable extension of part cloned into pSB1A30 with S/G on N-terminal or T/G on C-terminal; extension of linker and creating a friendly scar during parts fusion.
This was a biobrick compatable vector available in the lab to place our parts on.
constructed by former Ninfa lab member, Don Eun Chang
Mammalian expression vector (without a promoter) designed to add a eukaryotic RBS (Kozak) upstream (in-frame) and a nuclear localization tag, 6-histidine tag, and stop codon downstream (in-frame) of a protein coding insert. This method only works for Silver Standard (RFC 23) inserts.
* A Kozak-XbaI-NLS-6His-stop with SpeI sticky overhangs was inserted into the XbaI site. * Sequencing data confirms proper orientation of the inserted part. * XbaI site can accept any Silver Standard (RFC 23) protein coding region that is cut with XbaI/SpeI. * Single-cut vectors can easily re-ligate: It is highly recommended that the user dephosphorylates the XbaI-cut vector prior to ligation. * Insertion of a XbaI/SpeI insert is non-directional: It is high recommended that the user selects at least 3 colonies after ligation check (vi sequencing or some other method) for proper orientation of the insert.
MCS: SpeI BstXI EcoRI PstI EcoRV BstXI NotI XhoI
This integrative plasmid facilitates transformation of Synechocystis PCC6803 and similar species. It inserts gene of choice into the Synechocystis β-phellandrene synthase gene by homologous recombination.
Plasmid was purchased from open source plasmid repository Addgene.com. It was modified to be biobrick compatible. Formighieri, C., & Melis, A. (2014). Regulation of β-phellandrene synthase gene expression, recombinant protein accumulation, and monoterpene hydrocarbons production in Synechocystis transformants. Planta, 240(2), 309-324.
This vector is designed for Rhodobacter sphaeroides. You can transform it into Rhodoabcter spaherodies through conjugation.
To make it more flexible and easier to control, lacZ promoter and reporter have been removed.
This vector backbone is based on pMG170/pMG171. lac promoter and reporter have been removed from the original vector.
pSB1C3 + AF TALEN (K1480001) Target site
Backbone vector of pSB1C3 that contains a modification with a TALEN target site after the origin of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains a single TALEN target site (AF).
This plasmid backbone is based on pMA from Geneart. It has BioBrick prefix and suffix flanked on both sides by 50 bp of homology arms for insertion of the contained BioBrick into the 01000 locus of pCC2FosMB (BBa_K260001) by recombineering. The pMA-@CC2FosMB-01000bp backbone is available with BBa_K260015, which is composed of promoter-FRT-dhfr. If the PvuII fragment from BBa_K260005-BBa_K260015 is transferred by recombineering to the the 01000 locus of BBa_K260001-BBa_K260016, the intervening sequence between the FRT sites of BBa_K260015 and BBa_K260016 on the BBa_K260001 backbone will be 1,000 bp exactly. These plasmid backbones are similar and all allow recombineering-mediated transfer of the contained BioBrick (typically BBa_K260015) to the pCC2FosMB backbone BBa_K2600001. They differ in the homology arms, which determines the locus of insertion into pCC2FosMB. The bp numbers denote final inter-FRT distance as described above: BBa_K260004: pMA-CC2FosMB-00500bp BBa_K260005: pMA-CC2FosMB-01000bp BBa_K260006: pMA-CC2FosMB-02000bp BBa_K260007: pMA-CC2FosMB-05000bp BBa_K260008: pMA-RQ-CC2FosMB-10000bp
The 50 bp homologies were designed to delete 100 bp of BBa_K260001-BBa_K260016 and put the FRT site of BBa_K260015 (if used with the present backbone BBa_K260005) at a defined distance from the FRT site of BBa_K260016.
BBa_K260001 (pCC2FosMB) 3' homology FRT-01000bp
BBa_K260001 (pCC2FosMB) 5' homology FRT-01000bp
BsaI site removed from amplicillin resistance gene.
Erik Gullberg, Antonio Ascue Avalos, Erik Lundin
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.
p15A low to medium copy replication origin BBa_I50032
A plasmid backbone in which GeneArt often sends the constructions they're asked for, with kanamicine resistance, and restriction sites for SfiI, NarI, DraII, NcoI, Eco57I, and Col E1 origin.
pSB8S15(FRT) is a BioBrick standard vector with low copy pSC101ts replication origin (~5 copies per cell) and spectinomycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 repelication origin and propagates at 30???C but stop replicating at 42???C. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
Haissi Cui, Tilman Flock, Sebastian Gude, Christoph Hartlm??ller, Florian Praetorius, Jan Sch????rmann, Tobi Wauer, Philipp Wortmann
We designed a new screening systems based on the non-functional pSB1A10 plasmid. We improved its features for ??????in vivo?????? characterization of PoPS-based devices using fluorescent reporters . The plasmid still contains the Pbad arabinose-inducible induction system as a tunable input and eGFP as an internal standard for induction. However, we altered the reporter protein to mCherry. Furthermore we adjusted the BioBrick cloning site to allow cloning of additional parts independent from the Input/Output measurement. This screening plasmid is designed to be used with a second Arabinose inducible promoter BBa_I13453 which is not included in this part. The improved screening system is optimized for the evaluation of PoPS-based devices in fluorescence measurements. RFP which was known to contain an RNase restriction site was replaced by mCherry which combines good expression yield, short maturation times and an acceptable and well-characterized quantum yield. A unique challenge for the characterization of our switches is the expression of a corresponding signal independent of the input/output measurement. Thus, we moved the BioBrick cloning site resulting in the fluorescent reporter being inside the cloning site and giving the possibility to clone independent parts behind the reporter protein. To fully function our screening plasmid need the arabinose inducible promoter BBa_I13453 in front of the PoPS-based device to screen. Using a second arabinose inducible promoter, we were able to keep eGFP as an internal standard for the tunable input via the Pbad arabinose-inducible induction system. The two identical promoters ensure the same rate of induction for eGFP and the tested PoPS-based device. Thus, obtaining comparable screening results is easy. Unfortunately, this design implicates a minor disadvantage. Two cloning steps are needed to gain an functional construct for testing any PoPS-based device.
PoPS-based devices can be cloned into plasmid in front of the second reporter gene, whereas another independent sequence, like a signal, can be inserted behind the reporter genes.
Todd Eckdahl, Sachith Polpitiya Arachchige
The scaffold consist of four junctions, which allow to insert parts(promoters.RBS,Genes)between junctions A and Junction B,Junction B and Junction C or the Junction C and Junction D.
This is a geneart plasmid in which our antisense key part (BBa_K238005) is constructed.
Plasmid Backbone pSB1C95 for RFC 95 Open Sequence Initiative
Is the same pSB1C3 backbone but includes homing endonuclease sites before the Biobrick prefix and after the Biobrick suffix.
ampicillin + kanamycin resistant p15A vector (2ab vector)
Part is referred to internally as pBjh1601AK.
S. elongatus cyanobacteria sensor test rig
S. elongatus test vector for sensors. Has a cyanocitrus YFP coding sequence as a reporter. Comes with high copy number origin and kanamycin resistance
Redesigned by the BIOE44 teaching staff from pNS2-YFP.
Source plasmid is pNS2-YFP. pNS2-YFP is a plasmid made by Rachel Hood from Dr. Dave Savage's lab.
pSBBs2E: Empty backbone for integration into Bacillus subtilis lacA locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the lacA locus and can be selected with erythromycin + lincomycin (MLS). It has a ampicillin resistance for cloning in E.coli. This BioBrick is part of the LMU 2012 Bacillus BioBrick Box project: http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR. The clonging site also includes a rfp for selection in E.coli.
This part is an igem compatible version of the Bacillus subtilis vector pAX01
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).
Based on invitrogen's pYES2 plasmid. For mor information see the part information of pTUM100 (BBa_K801000).
pSB1C3 + BF and AF TALEN (K1480001) Target sites
Backbone vector of pSB1C3 that contains two modifications with a TALEN target after and before the origin of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains two TALEN target sites (AF+BF)
Gateway entry vector for gene cassette (L1L2)
Joy Jiao, Laura Deming, Adrian Slusarczyk
pGA4C5, Gibson assembly plasmid (bglBrick) with pLac-GFP insert
pSB1C3 + BF TALEN (K1480001) Target site
Backbone vector of pSB1C3 that contains a modification with a TALEN target site before the origin of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains a single TALEN target site (BF).
This plasmid backbone is based on pMA-RQ from Geneart. It has BioBrick prefix and suffix flanked on both sides by 70 bp of homology to pTetFlp (BBa_K260002) for tranfer of the contained BioBrick part by recombineering, for which the BioBrick needs to be excised with PvuII from the backbone. This plasmid backbone is available with BioBrick P_F3_zeoR_F3_RFP (BBa_K260017). The product of the above-described recombineering reaction BBa_K260002-BBa_K260017 is also available.
The 2x 70 bp homologies were designed to allow "pseudo-cloning" of a BioBrick from the present backbone '''pMA-RQ-@TetFlp''' to pTetFlp BBa_K260002 via recombineering using the PvuII fragment containing the BioBrick, where BioBrick prefix and suffix are kept untouched.
Geneart plasmid pMA-RQ, with 2x 70 bp from pTetFlp BBa_K260002.
pSB3C5 + MID + BF + AF TALEN (K1480001) Target sites
Backbone vector of pSB3C5 that contains three modifications with a TALEN target site on the center, after and before the origin of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains three TALEN target sites (MID+BF+AF)
The pGBKT7 vevtor expresses proteins fused to amino acids of the GAL4 DNA binding domain(DNA-BD).In yeast ,fusion proteins are expressed at high levels from the constitutive ADH1 promoter ;transcription is terminated by the T7 and ADH1 transcription termination signials. pGBKT7 also contains the T7 promoter, a c-Myc epitope tag, and a MCS. pGBKT7 replicates autonomously in both E.coli and S.cerevisiae from the pUC and 2μ ori, respectively. The vector carries the Kan for selection in E.coli and the TRP1 nutritional marker for selection in yeast. Yeast strains containing pGBKT7 exhibit a higher transformation efficiency than strains carrying other DNA-BD domain vectors.
suitable host strains:dh5α DH10 & other general and S.cerevisiae host strains:AH109 Y187 Y190 selectable marker:trp1
This palsmid backone is the standard GENEART vector used to synthesise iGEM parts. It is not a Biobrick compatible vector.
We used standard biobrick clonining sites to synthesise our Biobricks. So they should easily be cut with standard restriction enzymes used by iGEM.
Insertion Kit for Fusing Protein of Interest to Magnetosome Membrane
Consist of six fragments from magnetosome island of ''Magnetospirillum gryphiswaldense'' genome. Due to potential difficulty in introducing the large magnetosome formation operons into ''Azotobacter vinelandii'', a "template" consists of flanking sequences from '''mamXY, mamGFDC and mms6 operons''' except the mamC operon is designed for '''homologous recombination'''. Part containing this template ([http://parts.igem.org/Part:BBa_K1648002 K1648002]) together with another part containing mamC ([http://parts.igem.org/Part:BBa_K1648XXX K1648XXX]) is introduced into ''A. vinelandii'' genome via '''random integration''' before full operon sequences to be transformed into ''A. vinelandii''.
In order to form a homologous recombination template, we selected 250 bp of 5' and 3' ends from '''mamGFDC''', '''mamYXZ-ftsZ''' and '''mms6-mmsF-mms35-mms48''' ('''mamGC''', '''mamXY''' and '''mms6 operons''') except the mamC operon as flanking sequences. Fragments are joined via overlapping PCR.
Magnetospirillum gryphiswaldense MSR-1 v2, complete genome
T7 polymerase with an amber mutation coding gene is constructed after AraC protein(reversed sequence) and Pbad promoter which would be triggered by Arabinose. T7 polymerase can work well in the exsitence of SupD tRNA. Besides, the expression of the T7 polymerase can be regulated by RBS.
when cut this part for insert and ligate it into a vetor, the insert and vector may have the similar sizes which may disturb the ligation.
part registery 2009 distribution & PKU 2009 igem team
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.
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
{| border="1" |- |align="center"|'''Genotype''' |align="center"|'''Character''' |align="center"|'''Name''' |- |Top10/pT-TERM |So not express any repressors |align="center"|CR00 |- |Top10/pT-ARL4A0604 |Constitutively express ARL4A0604 |align="center"|CR01 |- |Top10/pT-ARL2A0203 |Constitutively express ARL2A0203 |align="center"|CR10 |- |Top10/pTet-ARL4A0604-ARL2A203 |Constitutively express ARL4A0604 and ARL2A0203 |align="center"|CR11 |}
pSB1A3_IntC - General Integration Plasmid with CmR
Mobilization protein (contains a 4bp deletion)
p15A origin, used in layered assembly of BglBrick parts
pSB4C3 is a low copy plasmid based with chloramphenicol resistance. It has a repA pSC101-derived replication origin. (Copy number ~10-12, Lutz and Bujard, 1997.) pSB4C5 is derived from the BioBricks vector scaffold BBa_I51001. However, the direction of the resistance marker and replication origin is not optimal.
BBa_I51001 BioBricks vector scaffold, pSC101 based origin and a version of the chloramphenicol rsistance cassette that has been cleaned of restriction sites.
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.
S. cerevisae test vector for sensors. Has a DasherGFP protein coding sequence which produces a GFP protein. Comes with high copy number origin, kanamycin resistance, and leucine yeast selection; can replace with low copy number origin if ordering from DNA 2.0
pSB4K15Iq is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and kanamycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. This vector carries a LacIq cassette on the backbone for tight repression of any lac promoter cloned into the cloning site.
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.
kanamycin + chloramphenicol resistant p15A vector (2ab vector)
pOP - protein expression vector compatible with RFC[25]
Ramona Emig, Julika Neumann, Lara Stuehn
This part contains the sequence for a plasmid backbone that is designed for efficient overexpression of proteins in ''Escherichia coli''. Usually, vectors that are optimized for protein overexpression are low- to medium-copy plasmids. In contrast to cloning purposes, this is sufficient for protein expression. When a certain copy number is reached, the expression machinery of the cell is saturated and a further increase will not result in higher expression yields. High copy plasmids rather result in lower expression rates because more metabolic capacity is shared for replication. Many ''E. coli'' strains optimized for overexpression carry a chloramphenicol resistance gene as a selective marker. Plasmids carrying the same marker are not suitable for protein expression for two reasons. First, double selection for successfully transformed colonies on a medium with two antibiotics is not possible. Second, there is no selective pressure which makes it an advantage for the cell to even keep the plasmid. The iGEM standard vector pSB1C3 was designed for cloning purposes and thus is provided with an origin of replication yielding high plasmid copy numbers. Moreover, it carries a chloramphenicol resistance gene as selection marker which makes it a tough task to use this vector for expression purposes. Using a modified version of pSB1C3 (plasmid map pIG15_001) for overexpression resulted in an unsatisfying protein amount. Working with a common expression vector increased the overexpression efficiency significantly. Obviously, it is a lot of work to clone every part you want to send to the registry into an expression vector and additionally into pSB1C3 which exhibits a completely different cloning site. For those reasons, this new vector backbone was added to the iGEM registry which is suitable for efficient protein overexpression and compatible for cloning parts derived from the registry in a standardized procedure. The new vector, called pOP ??? plasmid for Overexpression of Proteins - combines features needed for efficient overexpression of proteins with standardized elements derived from pSB1C3. Based on the commonly used expression vector pET22b+, some changes had to be applied to fit all the iGEM standards. First, one of the standard cloning sites had to be inserted instead of the original multiple cloning site. RFC[25] seemed to be the most suitable for protein expression purposes, because it allows the assembly of several coding sequences because the scar which is left between the sequences does not result in a frameshift or a stop codon. This is advantageous for expression of fusion proteins on the one hand and enables to genetically fuse the protein of interest to a specific tag for affinity purification on the other hand. Additionally, signal sequences can be added to cause secretion of the protein which is a commonly used tool for simplification of protein purification. Next, recognition sites for restriction enzymes which are used for the insertion of coding sequences had to be eliminated in the backbone, so those enzymes (namely AgeI, EcoRI, NgoMIV, NotI, SpeI, PstI and XbaI) remain single cutters. To allow sequence analysis in a standardized way, the binding sites of the primers VF2 and VR as they are used by the iGEM registry have been inserted in an appropriate distance to the insertion site. The final vector was designed theoretically based on pET22b+. Because the sites where adaptations had to be applied were distributed all over the vector, a multi-step mutagenesis approach was difficult. Instead, the vector was divided into five fragments. In the first step, two or three of the fragments were assembled by fusion PCR. The two resulting fragments were pieced together by Gibson Assembly. The resulting plasmid is shown in figure 1. Taken together, we designed a new plasmid for the overexpression of proteins that is compatible with iGEM cloning standards. We are happy to provide this new backbone to the iGEM registry and help future iGEM teams to reach high expression yields in an easy way.
The original sequence of pET22b+ was modified to make it compatible to iGEM standard cloning procedures. This includes the removal of recognition sites of several restriction enzymes as well as the insertion of RFC[25] as a multiple cloning site. Further details can be found on the main page.
The sequence is a modified version of the common expression vector pET22b+. Five fragments that were needed for the assembly of the whole part were synthesized by Integrated DNA Technologies.
pBR322 converted to BioBrick vector (formally BBa_I739201)
partial tetracycline resistance CDS (nonfunctional)
Streptomyces Shuttle Vetor Thiostrepton Resistance
This is a shuttle vector to be used with E. coli and Streptomyces. It has thiostrepton resistance.
This is a shuttle vector for Streptomyces.
This originated from the plasmid pRM5. Site-directed mutagenesis was used to remove specific restriction sites to make it biobrick compatible.
Vector with pSS Prefix B and Suffix C, confers kanamycin resistance, p15a origin of replication, constitutively expresses luxR and araC.
pSB4K5 is a low copy plasmid with kanamycin resistance. It has a repA pSC101-derived replication origin. (Copy number ~10-12, Lutz and Bujard, 1997.) It is derived from the BioBricks vector scaffold BBa_I51001.
BBa_I51001 vector scaffold, BBa_I50040 replication origin and BBa_P1003 antibiotic resistance marker.
pSB4A3-1 is a low copy plasmid with ampicillin resistance. It has a repA pSC101-derived replication origin. (Copy number ~10-12, Lutz and Bujard, 1997.) pSB4A3 has a terminator upstream of its MCS, which is oriented to prevent transcription from *inside* the MCS from reading out into any DNA outside the MCS.The orientation of the pBLA promoter driving the AmpR gene is such that relatively little transcription should be coming into the MCS from upstream. A second terminator (E.coli His operon-derived) is downstream of the MCS, again insulating the vector from transcription reading out of the MCS. Ideally, future versions of standard biobrick vectors would have terminators bracketing the MCS that were 100% efficient in terminating transcription both into and out of the MCS region.
Plasmid include T7 promoter, Histag and R9 peptide sequence
This plasmid has modified T7 promoter,RBS,His tag and nine Argenine signal peptide(R9 peptide). R9 peptide cause endocytosis of mammalian and plant cells and make them take not only R9 peptide but also Proteins combined to this peptide. you can correct and purify products of this promoter by using anti-His tag antibody.
pSB10K1 ??? contains mRFP cassette (J04450) and additionally terminators and flanking region from pSB1C3. The origin of replication has been replaced with ColA replicon with a copy number of 20 ??? 40 per cell. The cat gene has been replaced with kan cassette conveying kanamycin resistance.
Designed to be functional with pSB9S1, pSB3C7, and pSB6A3
pSB1C3 is the template with the ori and resistance replaced with those from pCOLA
Plasmid backbone used to put BioBricks into S. cerevisiae. Contains selection for uracil negative,ampicillin and LacZ.
Used mutagenesis to remove PstI at site 395???400 and XbaI at site 5294???5299. Rearrange EcoRI, XbaI, Pst1, and SpeI sites to be compatible with BioBrick standard.
Modified genomic sequence of pRS426 vector
Kanamycin resistant BAC with R6K origin, pGlpT 5' of cloning site
Kanamycin resistant BAC with R6K origin, glucose driven promoter 5' of cloning site. Can be maintained at higher copy in strains expressing pir. Referred to internally as pBacRJ2g
Andrew Halleran, Joseph Maniaci, Caroline Golino, Taylor Jacobs, John Marken, Elli Cryan, Panya Vij, Michael LeFew
We created a modified backbone that will allow users to quickly and easily create parts that can be integrated onto the E. coli genome. This backbone is designed as follows (where H1 refers to the first region homologous to the E. coli genome, and H2 refers to the second homologous region) In order to insert a part onto the genome, the user simply needs to put a part onto this backbone (between the prefix and suffix), and PCR up the region between (and including) H1 and H2. Users can put their parts onto this backbone with their preferred method of assembly. This part exhibits resistance to chloramphenicol and kanamycin. The parts integrated with this cassette will contain kanamycin resistance, and will be integrated in the galK region of the E. coli genome. Homologous regions modeled after those in Elowitz et al 2002.
iGEM Parts Registry. Homologous regions were modeled after those used in Elowitz, M. B., Levine, A. J., Siggia, E. D., & Swain, P. S. (2002). Stochastic gene expression in a single cell. Science, 297(5584), 1183-1186.
Mammalian test vector for actuators. Has a CMV promoter for constitutive expression in mammalian cells. Comes with high copy number origin and ampicillin resistance; can replace with low copy number origin and kanamycin resistance if ordering from DNA 2.0.
Developed by DNA2.0 from pcDNA3.1; IP free.
pSB4A15 is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and ??? antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
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).
pTUM102 yeast shuttle vector with pTEF2 promoter
E. coli test vector for actuators. Has a rhamnose inducible, glucose repressible promoter. Comes with high copy number origin and ampicillin resistance; can replace with low copy number origin and kanamycin resistance if ordering from DNA 2.0. Vector is licensed to DNA2.0 by Xbrane.
DNA2.0 licenses this plasmid from Xbrane and charges for its use.
Xbrane is has a patent on this plasmid, Patent Number: US008138324.
Psl2131 (for Synechocystis homologous recombination)
Kan/Amp resistant plasmid for Homogous recombination into Synechocystis 6803. Please note that this plasmid is only biobrick friendly and not compatible due to an EcoRI site present above the upstream homology region. To use this with biobrick compatible parts, assembly should be completed in a standard, highcopy plasmid such as Psb1C3 and then inserted into this backbone for transformation into Synechocystis using XbaI and PstI.
This plasmid has both Kan and Amp resistance.
This plasmid backbone was made by Bert Berla. It is a pUC19 derivitive.
pSB2K3 is a variable copy number plasmid carrying kanamycin resistance. The plasmid contains the F' replication origin and also the P1 lytic replication origin. Genes encoding proteins that are required to activate the P1 lytic replication origin are carried on the plasmid under the control of a lacI-regulated promoter. When grown in cells producing LacI the plasmid is maintained at very low copy (<10 per cell). When induced with IPTG the P1 lytic replication proteins are produced and the P1 lytic replication origin takes over, raising the copy number to very high levels (>100++ per cell). The copy control features of pSB2K3 are useful for cloning "difficult" parts while still being able to prepare large amounts of DNA. Additionally, pSB2K3 has terminators bracketing its MCS which are designed to prevent transcription from *inside* the MCS from reading out into the vector. The efficiency of these terminators is known to be < 100%. Ideally we would construct a future set of terminators for bracketing a MCS that were 100% efficient in terminating both into and out of the MCS region.
*** pSCANS via BNL *** The pSB2 series plasmids are based on the pSCANS-5 plasmid from John Dunn's group at Brookhaven National Laboratory. The pSCANS plasmids are introduced here.
MIT SBWG modification of pSCANS-5 from BNL
Biobricked pCS2+ plasmid with HSP70 promoter for frogs and chicken
This plasmid is a biobricked version of the plasmid pSC2+(K812000)with HSP70 promoter. This plasmid is made of a bacterial ampicilin resistant backbone. The Biobrick site is surrounded by a 5'UTR expression enhancer, and a poly-A tail with a SV40 3' UTR that enhance the stabilisation of the mRNA and the expression of the protein. To use this plasmid, you simply have to insert your protein between the EcoRI and PstI site. It is better to add a kozac sequence in front of your gene to enhance its expression. This plasmid contains the standard registry insert J04450 as a negative cloning control.
CMV of pCS2+ (K812000) was replaced by HSP70 using the double digestion SalI and HindIII. Then J04450 was replaced by sfGFP using the Biobrick K812031 with the double digestion EcoRI and PstI.
The plasmid template is from K812000, The constitutive CMV promoter was replaced by Heat Shock Promoter 70, and sfGFP is from K812031. HSP70 is from: Love, N.R. et al., 2011. pTransgenesis: a cross-species, modular transgenesis resource. Development (Cambridge, England), 138(24), pp.5451???8
pBS1C: Bacillus subtilis vector, amyE integration, CAM-resistance
This vector integrates into the amyE-site of Bacillus subtilis via double recombination. The two recombination sites flank a standard BioBrick multiple cloning site (with RFP-cassette), as well as a chloramphenicol resistance cassette (5 ??g/ml). The backbone does not contain the restriction sites EcoRI, XbaI, SpeI, PstI, NgoMIV nor AgeI. The vector is replicative in Escherichia coli and provides resistance to ampicillin (100 ??g/ml). Before transformation into B. subtilis, this vector should be linearized with ScaI.
This is one of three empty vectors with compatible integration loci and resistance cassettes to allow combination of up to three constructs of choice in one single strain.
pSB4A50: Low copy BioBrick cloning/expression backbone carrying Amp resistance
Absence of illegal sites in Vaccinia virus TK gene
Uses this typical vector as a substitute for Biobrick vectors to enable easier selection by blue white screening.
Obtained directly from commercial sources and used specifically to enable insertion of BBa_K408000.
pSB413-GPD: HIS3-selectable S. cerevisiae Expression Vector (RFC 10)
This is a RFC10 standard yeast expression vector adapted from the widely used plasmid pSB413-GPD. The MCS of p413-GPD was replaced with the standard BioBrick prefix and suffix and an illegal PstI restriction site near the HIS3 promoter of p413-GPD was removed. In the resultant plasmid, pSB413-GPD (5828 bp), the BioBrick prefix and suffix is situated between the strong constitutive yeast GPD promoter (TDH3) and CYC1 terminator allowing for facile cloning and expression of translational units in yeast. Standard VF2 and VR primer binding sites are included and M13 primer binding sites bracket the promoter and terminator. The plasmid has a ColE1 origin and ampicillin resistance for replication in E. coli. It also contains a yeast CEN/ARS and HIS3-selectable marker to provide high fidelity maintenance and low copy number in S. cerevisiae.
Homologous stable integration for nifH promoter T7 expression system Tookit
Seak Chi U, Chan Wai Lun, CHIU, Yee Ting
Platform for terminators analysis under the control of T7 promoter
We used this part to test terminators: the two subsequent proteins allow to quantify the termination efficiency as the ratio between Venus's and Cherry's fluorescence. The vector is pET21b, that contains lacI, t7 promoter and ampicillin resistance gene. The two fluorescent proteins have partially overlapping emission spectra, thus the fluorimetric measures should be taken with these parameters, to avoid interference between the two: Venus excitation: 485nm, Venus emission: 528nm Cherry excitation: 528nm, Cherry emission: 615nm We inserted the prefix-suffix linker between the two proteins, to allow terminator characterization.
Using and modifying this construct is important to remember that the two proteins have strong N-terminal homology, especially designing primers. For any future improvement, maybe it would be better two use two different fluorescence proteins, because these two has partially overlapping emission spectra and different folding kinetics. Using and modifying this construct is important to remember that the two proteins have strong N-terminal homology, especially designing primers. For any future improvement, maybe it would be better two use two different fluorescence proteins, because these two has partially overlapping emission spectra and different folding kinetics.
It's made from a Roberta Lentini's construct (from Mansy lab) that consist of pET21b with the two proteins and a 20bp linker between them. That construct was mutated two times to eliminate illegal restriction sites; prefix-suffix linker was added by insertion PCR.
pSB1AK3 is a high copy number plasmid carrying ampicillin and kanamycin resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell).
pSB4K15(FRT) is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and kanamycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
spectinomycin resistant pUC vector referred to internally as pBca9523.
pGA1C3, Gibson assembly plasmid (bglBrick) with pLac-GFP insert
BsaI site removed from amplicillin resistance gene. Contains stuffer piece of GCATGCATGCAT to be removed when cloning parts.
pSB6A3 - contains mRFP cassette (J04450) and additionally terminators and flanking region from pSB1C3. The origin of replication has been replaced with ColE1 replicon derived from pBR322 with a copy number of approximately 40 per cell. The cat gene has been replaced with ampR cassette conveying ampicillin resistance.
Designed to be stable with pSB9S1, pSB10K1, and pSB3C7
Original sequence used was pSB1C3, replacing the ori and resistance cassette with those from pET
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.
The ampicillin resistance marker needed to be eliminated.
Austin Che constructed this plamid from pSB1AT3-P1010.
Verifiction forward (VF2) primer binding site
Golden brick version of pSB1K3 plasmid for Golden Gate and Golden Brick cloning
This is a modified version of pSB1K3 from the registry with the J4450 negative control. The biobrick sites are modified to incorporate a BsaI restriction site that enables the use of golden gate cloning and golden brick cloning to assemble full contructs in a single shot of digestion/ligation. The complete protocol of the goldengate can be find in the original paper: Engler C, Gruetzner R, Kandzia R, Marillonnet S (2009) Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes. PLoS ONE 4(5): e5553. doi:10.1371/journal.pone.0005553 The Golden Brick assembly is an invention of the iGEM Evry 2012 Team. A complete description of the procedure can be find on our website: http://2012.igem.org/Team:Evry/GB
This part is a derivation of the pSB1K3 plasmid from the registry.
BioBrick integrative vector targeting aspA locus
It has been derived from BBa_K300000 base vector by exciding the default integration guide (attP of phi80 phage) and ligating aspA ( BBa_C0083 ) as guide.
It can be propagated in strains with pir or pir-116 locus. Integration works only in host strains with an intact aspA genomic locus.
BBa_K300000 specialized to target aspA locus.
pIL253-mRFP Biobrick Vector for L.lactis
This plasmid can be used to transform biobricks in L. lactis. Moreover it shuttles between L. lactis and B. subtilis. It contains mRFP Biobrick Prefix and Sufix. And it has an erythromycin resistance.
Cut out the multiple cloning sites of pIL253 also removed illegal restriction sites of EcoRI and XbaI
Yes, Derived from pIL253. Provided by molGen at the university of Groningen.
Light based screening plasmid backbone - pSB1Pc
Screening techniques available presently are all negatively related. The disadvantages of antibiotics are: 1. Principle of Negative Selection 2. Transformants carry a small but unnecessary risk of horizontal gene transfer to environmental microbes 3. Metabolic burden on the cells that significantly reduces the production of the desired protein Therefore, a novel selectable marker is needed which confers metabolic ???advantage??? to the transformants. This plasmid carries the gene proteorhodopsin.
Source : pSB1C3 is modified. Proteorhodopsin replaces the antibiotic gene.
Platform for terminators analysis under the control of tac promoter
This is the twin construct of BBa_K731700, with the same two subsequent fluorescent proteins, with T7 promoter replaced with Ptac. This construct allows to test possible differences in terminator efficiency due to different promoter and thus polymerase. We tested both constructs in the E.Coli strain BL21pLysS, that has both polymerases.
The presence of the promoter cannot be tested with screening digestion, as it has more or less the same T7 promoter`s length. Anyway the screening in amplification strains is much more easy: the right colonies will be intensely red.
The Ptac promoter has been inserted with a PCR, the sequence of the promoter has been taken from literature, while the original plamid is pET21b
pSB4S15(FRT) is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and spectinomycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
Vector with pSS Prefix C and Suffix D, confers kanamycin resistance, p15a origin of replication, constitutively expresses luxR and araC.
This plasmid backbone is based on pMA from Geneart. It has BioBrick prefix and suffix flanked on both sides by 50 bp of homology arms for insertion of the contained BioBrick into the 02000 locus of pCC2FosMB (BBa_K260001) by recombineering. The pMA-@CC2FosMB-02000bp backbone is available with BBa_K260015, which is composed of promoter-FRT-dhfr. If the PvuII fragment from BBa_K260006-BBa_K260015 is transferred by recombineering to the the 02000 locus of BBa_K260001-BBa_K260016, the intervening sequence between the FRT sites of BBa_K260015 and BBa_K260016 on the BBa_K260001 backbone will be 2,000 bp exactly. These plasmid backbones are similar and all allow recombineering-mediated transfer of the contained BioBrick (typically BBa_K260015) to the pCC2FosMB backbone BBa_K2600001. They differ in the homology arms, which determines the locus of insertion into pCC2FosMB. The bp numbers denote final inter-FRT distance as described above: BBa_K260004: pMA-CC2FosMB-00500bp BBa_K260005: pMA-CC2FosMB-01000bp BBa_K260006: pMA-CC2FosMB-02000bp BBa_K260007: pMA-CC2FosMB-05000bp BBa_K260008: pMA-RQ-CC2FosMB-10000bp
The 50 bp homologies were designed to delete 100 bp of BBa_K260001-BBa_K260016 and put the FRT site of BBa_K260015 (if used with the present backbone BBa_K260006) at a defined distance from the FRT site of BBa_K260016.
BBa_K260001 (pCC2FosMB) 5' homology FRT-02000bp
BBa_K260001 (pCC2FosMB) 3' homology FRT-02000bp
pBS2E: Bacillus subtilis vector, lacA integration, MLS resistance
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the lacA locus and can be selected with erythromycin + lincomycin (MLS). It has a ampicillin resistance for cloning in E.coli.
Compatible with pBs1C and pBs4S in one strain.
This backbone is a BioBricked version of the B. subtilis vector pAX01. This BioBrick is part of the LMU 2012 igem project [http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks '''''Bacillus''B'''io'''B'''rick'''B'''ox] Reference: [http://www.ncbi.nlm.nih.gov/pubmed/11274134 H??rtl ''et al.'']
This plasmid backbone is the same as BBa_K245004 except that it contains His tag inserted between multiple-cloning site and stop codon in suffix instead of between start codon and multiple-cloning site in prefix as in case of BBa_K245004.
Using overlapping primers and PCR ligation a fragment containing modified prefix, ccdB domain and modified suffix with His tag inserted between multiple-cloning site and stop codon was first obtained. The fragment was then ligated in pSB1A2 vector cut with EcoRI and PstI
The basis for BB-NIC-II-HisC is vector pSB1A2. Modified prefix and suffix are of synthetic origin. ccdB originates from part BBa_P1010.
This part allows a user to quickly and easily screen site specific methylating proteins for activity and specificity.
We worked to make the assay clear and as friendly to work with biobricks as possible.
Insulated vector based on pSB1C3. Uses B1010 transcriptional terminator upstream of BBa prefix and B0025 double transcription terminator downstream of BBa suffix.
Low copy BioBrick expression backbone carrying Kan resistance
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Büscher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schäfer, Carolin Schmelas, Silvan Schmitz, Max Waldha
pSB4S15Iq(FRT) is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and spectinomycin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. This vector carries a LacIq cassette on the backbone for tight repression of any lac promoter cloned into the cloning site.
Artificial Yeast Chromosome (Ura3 & Amp)
This part serves as a foundational contribution to expand the registry of yeast components. It is an easy to use artificial yeast chromosome with an auxitrophic uracil selection method for yeast expression, as well as an ampicillin selection for propagation in E.coli. The part was modified for biobrick compatibility from a working pRS316 plasmid containing a Gal1/10 bidirectional promoter which was removed through restriction digest. In addition to the biobrick prefix and suffix, the backbone contains Kpn1 and Sac1 restriction sites the flank the prefix and suffix respectively. Yeast transformants can be selected by using a uracil drop out medium and E.coli transformants may be selected using ampicillin resistance.
The plasmid backbone contained a Gal1/10 bidirectional promoter between the EcoR1 and BamHI sites on the pRS316 plasmid. The plasmid had to be modified for biobrick compatibility by using the HindIII and SacI sites to digest and ligate a gene fragment with the corresponding HindIII and SacI sites, but which also contained the biobrick prefix and suffix in the correct orientation.
The part was modified for biobrick compatibility from a working pRS316 plasmid containing a Gal1/10 bidirectional promoter which was removed through restriction digest.
pRS313 yeast shuttle vector with Biobrick MCS
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.
pSB1C3F-Vector pSB1C3 with RFP-cassette in Freiburg Standard
To allow the possibility to save some nucleotides of primer-overhangs, you can use this vector. The two AgeI-restriction sites from the original RFP-cassette have been removed. The cassette is flanked by the modified Freiburg standard (http://2012.igem.org/wiki/images/b/b9/LMU-Munich_2012_Our_Freiburg_standard_FusionPrefix.pdf) that includes an optimal ribosome binding site between XbaI and NgoMIV restriction sites.
Two AgeI-restriction sites have been removed by two subsequent quikchange site-directed mutagnesis reactions. The RFP-cassette was PCR-amplified with the overhangs: prefix: GAATTCCGCGGCCGCTTCTAGATAAGGAGGAACTACTATGGCCGGC suffix: ACCGGTTAATACTAGTAGCGGCCGCTGCAGT
pSB1C3; the RFP-part is originally from Discosoma sp.
Streptomyces Shuttle Vetor Apra Resistance
This is a shuttle vector to be used with E. coli and Streptomyces. It has apra resistance.
This originated from the plasmid pSET152. Site-directed mutagenesis was used to remove specific restriction sites to make it biobrick compatible.
Gateway entry vector used to store Aar1 AB and CD parts.
This plasmid consists of the mcherry gene (encoding red fluorescence protein) inserted in the backbone of pAW50 vector. EcoRI-NotI-XbaI-NsiI restriction sites have been introduced followed by a linker sequence followed by the mcherry sequence. The use of these sites will allow users to introduce any desired gene of their choice to be fused with the linker which is already fused with the mcherry sequence. This way any protein can be qualitatively studied in Methanogens via red fluorescence. Note:- the users will have to introduce the RBS sequence and start codon, delete the terminator sequence of their gene while cloning.
Primer were designed in a way to introduce EcoRI-NotI-XbaI-NsiI sites before the linker.
E. coli and lactobacilli shuttle vector with chloramphenicol resistance
E. coli Lactobacilli shuttle vector pSBLb4C15-red pSBLb4C15 is a shuttle vector containing the ori pSH71 originating from the plasmid pJP059 which was taken from Lactococcus lactis.
pSMB_MEASURE: Promoter Measurement plasmid (mammalian)
Lars Velten, Simon Haas, Anne Rademacher, Hannah Meyer, Michael Bartoschek and Chenchen Zhu
pSMB_MEASURE (SMB is for Synthetic Mammalian Biology) should be used for promoter characterization in mammalian cells. pSMB_MEASURE contains a reference promoter, JeT[1], which is flanked by BBb_2 (Tom Knight) sites and can therefore be replaced by the promoter to be measured. JeT is ideal as a reference promoter for a variety of reasons. First, it has an intermediate expression strength; second, it is regulated by a wide variety of transcription factors and low levels of change in fluorescence among different conditions. Third, we want to pay tribute to its creators as pioneers in synthetic promoter research. We separated JeT's core promoter from its proximal promoter by a HindIII site; it can therefore be used for screening of synthetic proximal promoters or for modifiying the strength of a promoter by core promoter swapping (described in our wiki http://2009.igem.org/Team:Heidelberg/Project_Synthetic_promoters ). In addition, it contains a FRT site which will allow for stable integration into mammalian cells also containing a FRT site. Thus, it provides the possibility to characterize the promoter in a defined genome and in this way helps to avoid some of the challenges we identified for promoter characterization. For the same reason, it also contains a mammalian selection marker (hygromycine). For the generation of the plasmid, please see our Notebook. As a reporter gene, it contains GFP, which is followed by a SV40 mammalian terminator. We generated another plasmid pSMB_REFERENCE, which contains mcherry instead of GFP. It can be used for normalizations to transfection efficiency.
As a starting plasmid, we used pcDNA5/FRT (Invitrogen), as having this plasmid at hand was an immediate requirement for our project and we did not have the time to construct it from parts. We nevertheless highly modified it by performing two site-directed mutageneses to remove prohibited cutsites, we removed the CMV promoter it contained and inserted JeT, a well-described[1] synthetic promoter which we modified to contain a HindIII cutsite between the proximal and the core promoter for promoter screening. We also added full BBB_2 (Tom Knight) sites to it. Then, we synthesized it by Assembly PCR and inserted it via a MfeI and a PstI site. We then fixed eGFP plus a Kozak-sequence (eukaryotic RBS) into the plasmid backbone by cutting with PstI and BClI References: [1] Tornoe, J. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 297, 21-32 (2002).
plasmid: modified from pcDNA5/FRT (Invitrogen) as described below GFP: PCR from a plasmid containing eGFP-N1
pSB1A2 w/ "A" at -32 upstream from RNAII
pCDF-1b carries a T7 promoter and lac operator to control transcription, a replication origin derived from CloDF13, and streptomycin/spectinomycin antibiotic resistance (SmR). It also encodes an N-termi- nal His???Tag?? sequence followed by an enterokinase (Ek) cleavage site and an optional C-terminal S???TagTM sequence. Unique sites are shown on the circle map. pCDF-1b is compatible with pET vectors (ColE1 origin), pRSF vectors (RSF1030 replication origin), and pACYC derived plasmids (P15A replica- tion origin) carrying compatible antibiotic resistance markers. Sequencing can be performed using the T7 Promoter Primer (Cat. No. 69348-3) and AS S???Tag 18mer Primer (Cat. No. 71262-3) or T7 Terminator Primer (Cat. No. 69337-3). (Taken from Novagen)
This is the plasmid backbone to which BBa_K512002 is inserted to by NcoI and Acc65I.
pSB3T5 is a low to medium copy plasmid based with tetracycline resistance. It has a p15A-derived replication origin. pSB3T5 is derived from the BioBricks vector scaffold BBa_I51001.
BBa_I51001 BioBricks vector scaffold, p15A based origin and a version of the tetracycline resistance cassette that has been cleaned of restriction sites.
pSB4A15 is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and ampicillin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. The resistance cassette is flanked by FRT sites (BBa_J61020) which allows for subsequent removal of the resistance cassette from the chromosome by the FLP recombinase. The pSB8x15 series are especially suitable for carrying the FLP recombinase, since they can be removed by incubation at 42??C.
The plasmid pAW42-gs(ugaiGEM2)consists of the gs gene inserted into the backbone of pAW42-vector. The gs gene encodes for geraniol synthase enzyme. This vector has puramycin marker for selection in Methanogens and ampicilin marker for selection in E. coli.
Codon optimized for expression in Methanococcus.
The gs gene was obtained from Ocimum basilicum.
Plasmid backbone pSB1A3 carrying the arabinose induction system
pBS1C, Bacillus subtilis vector, CAM-resistance, amyE-integration
amyE-integration, chloramphenicol resistance. ampicillin resistance and origin of replication for Escherichia coli. Lacks EcoRI, XbaI, SpeI, PstI, AgeI, NgoMIV - restriction sites (except for the MCS, obviously, which contains the RFP-cassette).
empty, integrative vector for Bacillus subtilis. Compatible with pBS2E and pBS4S
This plasmid backbone is based on pMA-RQ from Geneart. It has BioBrick prefix and suffix flanked on both sides by 50 bp of homology arms for insertion of the contained BioBrick into the 10000 locus of pCC2FosMB (BBa_K260001) by recombineering. The pMA-RQ-@CC2FosMB-10000bp backbone is available with BBa_K260015, which is composed of promoter-FRT-dhfr. If the PvuII fragment from BBa_K260008-BBa_K260015 is transferred by recombineering to the the 10000 locus of BBa_K260001-BBa_K260016, the intervening sequence between the FRT sites of BBa_K260015 and BBa_K260016 on the BBa_K260001 backbone will be 10,000 bp exactly. These plasmid backbones are similar and all allow recombineering-mediated transfer of the contained BioBrick (typically BBa_K260015) to the pCC2FosMB backbone BBa_K2600001. They differ in the homology arms, which determines the locus of insertion into pCC2FosMB. The bp numbers denote final inter-FRT distance as described above: BBa_K260004: pMA-CC2FosMB-00500bp BBa_K260005: pMA-CC2FosMB-01000bp BBa_K260006: pMA-CC2FosMB-02000bp BBa_K260007: pMA-CC2FosMB-05000bp BBa_K260008: pMA-RQ-CC2FosMB-10000bp
The 50 bp homologies were designed to delete 100 bp of BBa_K260001-BBa_K260016 and put the FRT site of BBa_K260015 (if used with the present backbone BBa_K260008) at a defined distance from the FRT site of BBa_K260016.
Geneart plasmid pMA-RQ, with 2x 50 bp from pCC2FosMB K260001.
BBa_K260001 (pCC2FosMB) 5' homology FRT-10000bp
BBa_K260001 (pCC2FosMB) 3' homology FRT-10000bp
ampicillin resistant pUC vector part referred to internally as pBjk3057
pSBBs0K-Pspac (replicative Bacillus subtilis expression vector; IPTG inducible
This part is an expression vector for Bacillus subtilis. It is replicative both in E.coli and B.subtilis. It has a ampicillin resistance for cloning in E.coli and kanamycin resistance for selection in B. subtilis. The multiple cloning site is downstream of a Pspac promoter, that is inducible with IPTG. (Isopropyl-β-D-thiogalactopyranosid). This BioBrick is part of the LMU 2012 igem project Bacillus BioBrickBOX http://2012.igem.org/Team:LMU-Munich/Bacillus_BioBricks
We removed any forbidden restriction sites by site-directed mutagenesis PCR. The clonging site also includes a rfp for selection for cloning in E.coli. It can be used as an inducible expression vector in B. subtilis.
This part is an igem compatible version of the Bacillus expression vector pDG148
pSB1A30: High copy BioBrick cloning/expression backbone carrying Amp resitance
pDCPKO - Integrative plasmid for B. subtilis
It's an integrative plasmid for B. subtilis, biobricked with AmyE 5' and 3' sequence.
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).
pSB3C5 + Before and MID TALEN (K1480001) Target site
Backbone vector of pSB3C5 that contains a double modification with a TALEN target site before the origin of the plasmid and other on the center (not in the origin). It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains a two TALEN target site (Bef and Mid).
Plasmid enabling Yarrowia lipolytica biobrick
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).
pTetFlp. [anhydrotetracycline] -> FLP(F70L)
This plasmid is based on pSC101 and contains TetR as well as the F70L variant of FLP recombinase under tet-promoter control. Therefore anhydrotetracycline can be used to induce FLP(F70L) expression. The pSC101 origin of replication is temperature sensitive due to a mutation of repA. Permisive temperature is 30 degC. 37 degC should be avoided as the plasmid can not replicate.
E. coli test vector for sensors. Has a Gemini protein coding sequence which produces a LacZalpha-GFP fusion protein. Comes with high copy number origin and ampicillin resistance; can replace with low copy number origin and kanamycin resistance if ordering from DNA 2.0.
Gemini coding sequence sourced from Dr. Drew Endy and Martin et al., PlosOne 2009. The rest of the vector is sourced from DNA2.0.
Low to medium copy Lambda Red recombineering compatible plasmid
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.
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).
pSB4A5 is a low copy plasmid with ampicillin resistance. It has a repA pSC101-derived replication origin. (Copy number ~10-12, Lutz and Bujard, 1997.) It is derived from the BioBricks vector scaffold BBa_I51001.
See http://openwetware.org/wiki/Synthetic_Biology:Vectors/pSB%2A%2A5_design
BBa_I51001, pSC101 origin and a new version of ampR stripped of restriction sites.
ampicillin resistant p15A vector with dapD
ampicillin resistant p15A vector with dapD referred to internally as pBjk3066A
Protein fusion vector (Silver lab standard)
standard biofusion plasmid containing ccdB death gene and chlorenphenicol resistance
This integrative plasmid facilitates transformation of Synechocystis PCC6803 and similar species. It inserts gene of choice into the Synechocystis psbA2 gene by homologous recombination.
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.
pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). For the version of this plasmid with a CcdB insert, see BBa_P1010.
Silent g->a mutation found during sequencing
pSB1A3_IntC6803 - Synechocystis sp. PCC 6803 Integration Plasmid with CmR
Inducible plasmid. Allows the control over copy number of plasmid. Maintain at a low copy number during screening and then before DNA purification induce for higher copy numbers and yields.
Episomal stable eucaryotic expression vector (pDSG)
Plasmid for genomic integration in HEK293 cells
pSB2K3-derived BioBrick plasmid free of many restriction sites
Derived from pSB2K3 with many restriction enzyme sites removed by site-directed mutagenesis (~2 dozen mutations). Plasmid is useful when there is a need to cut with a variety of different restriction enzymes. Most symmetric cutters have been removed, and some non-symmetric cutters have also including AarI, AcuI, BbsI, BbvCI, BciVI, BsaI, BsmBI, EarI, N.BstNBI, and SgrAI. Works in exactly same way as pSB2K3. See <bb_part>pSB2K3</bb_part> for more information. Note that the VF2 site has a single base mutation in the middle of it. However, VF2 has been used successfully as a primer for PCR with this plasmid.
pSB1A3-1 is a high copy number plasmid carrying ampicillin resistance. The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell). pSB1A3 has terminators bracketing its MCS which are designed to prevent transcription from *inside* the MCS from reading out into the vector. The efficiency of these terminators is known to be < 100%. Ideally we would construct a future set of terminators for bracketing a MCS that were 100% efficient in terminating both into and out of the MCS region. A fully annotated Genbank file is attached below.
pSB1C3 + MID and AF TALEN (K1480001) Target sites
Backbone vector of pSB1C3 tha contains a modification with a TALEN target site on the center and after the origin of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems. It contains two TALEN target sites (MID+AF)
ampicillin resistant split ColE2 vector. Requires expression of RepA to replicate. Referred to internally as pBjk2992.
Multi-host vector pTG262 converted to BioBrick vector
biobricked version of pTG262 Plasmid has a multi host replication origin and is competant in range of gram positive and gram negative bacteria.
We kindly thank Dr Mike Gasson & Dr. Claire Shearman, of the Institute of Food Research in Norwich for generously donating the pTG262 vector.
Susanna Zucca, Federica Sampietro, Paolo Magni
This part is identical to pSB1A2, but it does not have the Prefix and Suffix. It can be useful to specify the sequence of new vectors constructed with BioBrick parts, such as BBa_K300001 .
No changes have been made to pSB1A2 sequence.
pSB4C50: Low(?) copy BioBrick cloning/expression backbone carrying Cm resistance
pSB1T30: High copy BioBrick cloning/expression backbone carrying Tet resitance
Reconstructed pSB1C3 for plant transfection
2AB Construction Plasmid with Chloramphenicol and Kanamycin resistance. ---- This part is in BBb Format. It is flanked by BglII and BamHI sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Requires RepA of ColE2 to be provided in trans.
The broad-host-range shuttle vector pPMQAK1 is a BioBrick-compatible plasmid that provides ampicillin and kanamycin/neomycin resistance (Genbank GU933126). As pPMQAK1 contains part of the pSB1AK3 plasmid it contains the same flanking terminators, and it is possible to use the VF2 (BBa_G00100) and VR (BBa_G00101) primers for colony PCR and sequencing. It was originally developed to allow for the use of BioBrick constructs in cyanobacteria (Huang etal 2010). But because of its RSF1010-derived broad-host-range replicon, it is expected to replicate successfully in most gram negative bacteria (Meyer 2009). Plasmids with a RSF1010-derived replicon, which belong to the IncQ group, have been found to have a copy number of 10 in Escherichia coli (Frey and Bagdasarian 1989), and from 10 (Marraccini etal 1993) to 30 (Ng etal 2000) per cell in the cyanobacterium Synechocystis sp. PCC 6803. Practical notes pPMQAK1 can be transformed into competent E. coli as preferred, but appears to give a lower number of transformants as compared to other cloning vectors (such as the pSB1***-series). The relatively low copy number also means that plasmid prep yields will be low. Further, it is often difficult to completely digest all plasmid, as judged by agarose gel electrophoresis. Conjugation (triparental mating) can be used to transfer pPMQAK1 into cyanobacteria as it carries the required mobilization genes and an oriT (origin of transfer, or bom-site) that enable conjugative transfer with the help of an additional conjugal plasmid. More info about engineering cyanobacteria, conjugation and detailed protocols can be found in the dedicated Methods in Enzymology chapter (Heidorn etal 2011). References Frey,J. and Bagdasarian,M. (1989) The molecular biology of IncQ plasmids. In Thomas,C.M. (ed.), Promiscuous Plasmids of Gram-negative Bacteria. Academic Press, London, pp. 79???94. Heidorn, T., Camsund, D., Huang, H.H., Lindberg, P., Oliveira, P., Stensjo, K. and Lindblad, P. Synthetic biology in cyanobacteria engineering and analyzing novel functions. Methods Enzymol, 497, 539-579. Huang, H.H., Camsund, D., Lindblad, P. and Heidorn, T. (2010) Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res, 38, 2577-2593. Marraccini, P., Bulteau, S., Cassier-Chauvat, C., Mermet-Bouvier, P. and Chauvat, F. (1993) A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis. Plant Mol Biol, 23, 905-909. Meyer, R. (2009) Replication and conjugative mobilization of broad host-range IncQ plasmids. Plasmid, 62, 57-70. Ng, W.O., Zentella, R., Wang, Y., Taylor, J.S. and Pakrasi, H.B. (2000) PhrA, the major photoreactivating factor in the cyanobacterium Synechocystis sp. strain PCC 6803 codes for a cyclobutane-pyrimidine-dimer-specific DNA photolyase. Arch Microbiol, 173, 412-417.
As cloning by plasmid preparation and subsequent digestion/ligation was problematic due to low copy number and difficulties in digesting the RSF1010-derived part from pAWG1.1, pPMQAK1 was made using PCR and subsequent digestion/ligation.
pPMQAK1 was constructed by ligating a PCR product from the pAWG1.1 plasmid, corresponding to the RSF1010 replicon, with a PCR product from the pSB1AK3 plasmid, corresponding to the BioBrick cloning site with flanking functional sequences plus the ampicillin and kanamycin resistance cassettes.
Plasmid Backbone for BBb standard of biobrick. Used for 2ab assembly
E. coli Lactobacilli shuttle vector pSBLb4C15
Insuring it followed biobrick standard 10
Ori from plasmid pJP059 inserted into pEL4C15 with ori removed
pSWK oriTRP4; [CmR]; oriVR6K; lambda_attP
This vector has the conditional origin of replication oriVR6K and can be maintained only in strains expressing the pir gene. It carries the origin of transfer of RP4 and can thus be used in conjugation assays in a donor cell expressing the RP4 conjugation machinery. It also carries the attP recombination site of the lambda phage in order to permit site specific integration at the attB site of E.coli chromosome mediated by the lambda integrase (Valens et al., 2004).
attP recombination site of the lambda phage was PCR amplified with oligos 5'-TGGAGCTCAAATCAAATAATGATTTTATTT-3' and 5'-GCTGGAGCTCCATGGTACGCGTGCTAGAGGCA-3', and cloned at the SacI site of pSW23T (Demarre et al., 2005). The multiple cloning site was then replaced with the BioBrick strandard restriction sites through inverse PCR with oligos 'GCTTCTAGAGTACTAGTAGCGGCCGCTGCAGGCGGTGGAGCTCAAATCAAATAATG' and 'AGTACTCTAGAAGCGGCCGCGAATTCTATCAAGCTTATCGATACCGTCGACG'
This plasmid was constructed from pSW23T (Demarre et al., 2005)
Plasmid for construction of promoter basic parts. Insertion of a promoter element between XbaI and SpeI sites results in a RFP reporter while retaining the ability to do biobrick assembly. Part J61002 is the tet promoter variant of the plasmid.
PCR ca1020F/R on pSB1A2-I13521 (900bp, SpeI/PstI) Sub into pSB1A2-I13522 (SpeI/PstI) Product is pBca1020, will be an iGEM number ultimately ---- ca1020F Forward SpeI oligo for X-rbsRFP-P plasmid gcactACTAGTgaaagaggagaaatactagatggc ca1020R Reverse PstI oligo for X-rbsRFP-P plasmid ccggactgcagcggccgcttctagtatataaacg
pSB416-GPD: URA3-selectable S. cerevisiae Expression Vector (RFC 10)
Danielle Bauhan, Alexander Lacrampe, Joseph Lee, and Richard Anthony
This is a RFC10 standard yeast expression vector adapted from the widely used plasmid p416-GPD. The MCS of p416-GPD was replaced with the standard BioBrick prefix and suffix and an illegal PstI restriction site near the URA3 promoter of p416-GPD was removed. In the resultant plasmid, pSB416-GPD (5743 bp), the BioBrick prefix and suffix is situated between the yeast GPD promoter and CYC1 terminator allowing for facile cloning and expression of translational units in yeast. M13 primers flank the transcriptional unit. The plasmid has a ColE1 origin and ampicillin resistance for replication in E.coli. It also contains a yeast CEN/ARS providing high fidelity maintenance and low copy number in yeast.
Replacing the MCS with standard BioBrick prefix and suffix, removing illegal restriction sites and avoiding deleterious sequences in the promoter and terminator regions.
This plasmid is adapted to the BioBrick Standard from p416-GPD (Mumberg, D., M??ller, R. and M. Funk (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 156. pp. 119???22.)
The plasmid is designed to integrate a cloned insert into the B. subtilis 168 chromosome at the sacA locus. The user inserts the fragment inside the MCS whi
Plasmid with BioBrick 3.0 prefix and suffix
we had to design the new MCS the way that would not create stop codon while translating out protein.
BBa_K592201 is a BioBrick friendly vector with low to medium copy p15A replication origin (BBa_I50032) and chloramphenicol 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 chloramphenicol 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 resistance marker from the pKD3 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.
The P22 late terminator come from the genome of the Salmonella phage P22 (GenBank: AF217253.1, 19662..19702), and the chloramphenicol resistance marker flanked by FRT sites comes from the pKD3 plasmid (Datsenko and Wanner, 2000).
ThrC integration backbone for B. subtilis
Replication of tour bioBrick can via integration in B. subtilis locus. One of these loci is the ThrC locus. This backbone is based on the plasmids pdg1664 and K823023. The AmyE loci from K823023 were replaced with the ThrC loci from pdg1664. Along with this exchange, the chloramphenicol resistance gene was replaced with erythromycin resistant gene (wich was also found in the pdg1664). This biobrick can be replicated both in E. coli and in B. subtilis, where it intergrates into the genome.
pdg1664 contained an EcoRI and a XbaI restriction site. However these sites were situated around a locus. An option was to introduce the prefix, suffix and remove the other restriction site. Here is chosen to copy the loci and erythormycin resistance gene and use these to replace the AmyE intergration loci and chloremphencicol resistance gene from Bba_K823023.
This backbone comes from combining Bba_K823023 and pdg1664
Vector with pGA Prefix/Suffix, confers kanamycin resistance, p15a origin of replication, constitutively expresses luxR and araC.
Inserted luxR and araC to be constitutively expressed in the reverse direction on the vector.
Eukaryotic terminator vector (very similar to pSB1AK8)
BioBrick prefix partial (missing G at 3' end)
This is a high copy plasmid for use in assembly of BioBrick?? standard biological parts.
3 bases added after PstI site to allow making overlapping PstI/BstXI site
G418 selection yeast shuttle vector designed for BioBrick
George Chao, Misha Patel, Hannah Aho, Molly Swanson, Dana Morrone
A yeast/E. coli shuttle vector was designed with BioBrick prefix and suffix sites such that a yeast expression cassette could be inserted through basic BioBrick assembly. G418 was chosen instead of an auxotrophic marker for plasmid selection in yeast. To synthesize the overall plasmid backbone, PCR primers were designed to amplify five desired fragments (with 25-30 bp overlap) from different plasmids, which could be joined by Gibson Assembly: 1. BioBrick destination plasmid pSB1C3 containing the MCS and rep (pMB1). 2. BioBrick destination plasmid pSB1C3 containing the Chloramphenical resistance gene. 3. 2u Gibson Extract provided by the Schmidt-Dannert lab (University of Minnesota) containing the 2u ORI for yeast. 4. pkT127 provided by the Schmidt-Dannert lab containing G418 resistance genes and tTEF1. 5. ATCC plasmid provided by the Schmidt-Dannert lab containing the ADH1 promoter to drive the G418 R gene.
A number of mutations were directed so as to maintin the uniqueness of BioBrick cut sites.
This plasmid was assembled though Gibson assembly. Gibson assembly pieces were amplified from preexisting plasmid templates.
Inspired by the measkit BBa_I20260 . The RFP measkit template with swappable promoter part, taken from BBa_J61002 .
Non-standard suffix and prefix of BBa_J6100 is inherited.
pSB4A15 is a BioBrick standard vector with low copy pSC101 replication origin (~5 copies per cell) and ampicillin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22. This vector carries a LacIq cassette on the backbone for tight repression of any lac promoter cloned into the cloning site.
pSB1C3 + MID TALEN (K1480001) Target site
Backbone vector of pSB1C3 tha contains a modification with a TALEN target site on the center of the plasmid. It can be used as a target for the respective TALEN enzyme in DNA edition projects or processing information systems.
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 plasmid has modified T7 promoter,RBS and nine Argenine signal peptide(R9 peptide). R9 peptide cause endocytosis of mammalian and plant cells and make them take not only R9 peptide but also Proteins combined to this peptide.
Low copy protein fusion vector (Silver lab standard)
Silver Lab alias V0002. This is a modified biobrick vector (fusion brick vector) that contains the specified EcoRI, SpeI, XbaI, and PstI sites. The only difference is that the part sequence immediately follows the XbaI site and immediately precedes the SpeI site. This change allows in-frame fusions of protein coding regions to be made using standard biobricks assembly methods.
If the part starts with the nucleotides tc, then that part-containing plasmid must be grow in a DamI- strain to avoid methylation (and inhibition of digestion) at the XbaI site.
S. elongatus cyanobacteria actuator test rig
pSBBs4S: Empty backbone for integration into Bacillus subtilis thrC locus
This part is an empty backbone vector for the usage in Bacillus subtilis. It integrates in the thrC locus and can be selected with Spectinomycin cassette. It has a ampicillin resistance for cloning in E.coli. It originates from the Bacillus vector pDG1731.
This plasmid can be used as empty backbone vectors like pSB1C3 but is able to integrate in B.subtilis. The clonging site also includes a rfp for selection.
Transcriptionally insulated high copy BioBrick plasmid
pSB1A4 was constructed by adding double terminators ([BBa_B0015]) before the BioBrick prefix and after the BioBrick suffix in pSB1A3. This insulator blocks read-through transcription into BioBrick parts/ devices.
The double terminators were assembled from small smaller overlapping ssDNA oligos. The outer-most EcoRI and PstI sites were mutated while the inner-most BioBrick cut sites were restored.
Based upon pSB1A3; Includes two copies of [BBa_B0015] assembled from DNA oligos
Vector with pSS Prefix A and Suffix D, confers kanamycin resistance, p15a origin of replication, constitutively expresses luxR and araC.
pSB3K5 is a low to medium copy plasmid based with kanamycin resistance. It has a p15A-derived replication origin. pSB3K5 is derived from the BioBricks vector scaffold BBa_I51001.
BBa_I51001 BioBricks vector scaffold, p15A based origin and a version of the kanamycin resistance cassette that has been cleaned of restriction sites.
BBa_K300000 with RBS-RFP-TT between SpeI-PstI in the suffix
This is the integrative base vector BBa_K300000 with a mRFP1 gene with a strong RBS and a double terminator between the SpeI-PstI sites in the BioBrick suffix. See BBa_K300000 for detailed feature information. The RBS-RFP-TT is the SpeI-PstI fragment coming from BBa_J61002 .
BBa_K300000 (backbone) and BBa_J61002 (SpeI-PstI insert).
pBBR1MCS-5 -> broad-host-range cloning vector
The plasmid we designed is a modification of the pBBR1MCS-5 , this broad-host-range (bhr) vector has been designed to assist the genetic analysis in prokaryotes, specifically in Gram- bacteria that are naturally CmR (chloramphenicol resistant). The pBBR1MCS-5 vector is relatively small (4768bp), it has an extended multiple cloning site (MCS) with 15 different restriction sites and it possesses a gentamicin resistance gene. After modification, only 3 of the 15 retrictions sites are present (see design notes). The plasmid is mobilizable when the RK2 transfer functions is provided in trans and is compatible with IncP, IncQ and IncW group plasmids, as well as with ColE1- and P15a-based replicons. We used this plasmid to transfer genes from E. coli to R. etli. First we transformed E. coli S17 with this vector containing the genes or sequences that we wanted to transfer, then we did a conjugation between our transformed E. coli cells and Rhizobium etli CFN42
It was amplified the BBa_J04450 including the prefix and the suffix from the psb1t3 plasmid and adding apaI and sacI restriction sites at the ends of the sequence. After, a ligation by sticky ends was with the pBBR1MCS-5 the apaI and sacI enzimes, trying to eliminate as mouch as possible of the MCS??? restriction sites We use the sequence as reported by Kovach et al. (1995).
This is one of the antibiotic-resistant derivatives of the broad-host-range cloning vector pBBR1MCS.
The first vector of the Golden gate series (adapted from psb1c3) This vector expedites golden gate cloning with BBa_K560013, BBa_K560014 and BBa_K560015. To make this set of vector compatible with the standard biobricks, we have maintained PstI and XbaI recognition sites. Flanking these two sites are the cleavage and recognition sites of BsaI, a kind of Type IIS restriction endonuclease. This kind of restriction endonucleases cleaves outside their recognition sites thus creating different sticky ends. If you want to assemble four biobricks at a time, you can put the biobricks in order into corresponding vector by the digestion of PstI and Xbal. Then mix these assembled vectors and a backbone (PFUS4 from TALEN golden gate kit). Under the effect of ligase and Bsal, these biobricks will have sticky ends nose to tails and ligate in order in a single reaction.
IPTG inducible E. Coli Expression vector with RCF25 compatible MCS
pSB3C7 ??? contains mRFP cassette (J04450) and additionally terminators and flanking region from pSB1C3. The origin of replication has been replaced with P15A replicon with a copy number of 10-12 per cell. The cat gene has been maintained conveying chloramphenicol resistance.
Designed to be co-expressed with pSB9S1, pSB10K1, and/or pSB6A3
Sequence comes from pSB1C3 with the ori and resistance changed form that of pACYC.
pSB8K15 is a BioBrick standard vector with low copy pSC101ts replication origin (~5 copies per cell) and ampicillin antibiotic resistance marker, especially usable for Lambda Red recombineering in E coli. The pSC101ts is a temperature sensitive pSC101 repelication origin and propagates at 30???C but stop replicating at 42???C. The backbone sequence is based on pSB3T5, but the E coli His operon terminator BBa_B0053 has been replaced with the late terminator of the Salmonella phage P22.
This part is a phagemid. It functions as a normal plasmid when transformed into bacteria. But when helper phage M13KO7 infect this bacteria, it will produce progeny M13 phage. Phagemid pBluescript contains a f1 ori while M13KO7 helper phage's package signal is modified as knock-down. So after the infection of M13KO7, its genome can produce protein but these proteins will see pBluescript as their true ???genome???. As a result, the progeny phage???s genome will only contains pBluescript, so they cannot produce next generation by infection. We have removed some sequence on its MCS to create a linearized form of this phagemid.
Normally, a pBluescript contains EcoRI, PstI, XbaI and SpeI on its MCS. We have removed these sequence to create a linearized form of this phagemid.