Origin for Asaia and resistance to Kanamycin
Forward primer for sequencing/amplifying BioBrick parts (VF2)
Originally named VF2, primer binds upstream of the standard BBa_MCS on biobrick vectors.
This part contains a stop codon in all 6 reading frames. Useful for ensuring that translation does not proceed through this part.
F plasmid backbone with BioBrick sites removed, reverse
This is a (near) single copy origin derived from the F plasmid. The naturally occurring BioBricks sites were removed with an intent to preserve the amino acid and codon frequency where possible. Similar to the pSMART VC sequence from Lucigen http://www.lucigen.com/products/Cloning_Systems/CopyRight.html. It is intended to be combined with other vector components (antibiotic resistance marker, multiple cloning site) in order to form a BioBricks vector. Relevant information can be found at http://openwetware.org/wiki/BioBrick_Parts_for_Plasmid_Engineering. This part is a reverse copy of BBa_I50000.
sequence derived from F plasmid in GenBank Accession Number AP001918
ori2 (oriS) single copy replication origin
C to G mutation (eliminates XbaI site, noncoding region)
This part was amplified from pSB4A3-P1010.
pSC101 origin of replication which is supposed to have low copy number. This was the origin used in Michael Elowitz's repressilator and in Bujard's pZS4Int-1 vector with mutations introduced to eliminate a SpeI site. It is also the origin of replication in pSB4* series plasmids.
The replication origin boundaries were chosen quite liberally (i.e. they might include more sequence than is strictly necessary) because a different version of this origin (BBa_I50040) was shown to be non-functional.
T to C difference with canonical pSC101 vector sequence
G to C difference with canonical pSC101 vector sequence
C to T difference with canonical pSC101 vector sequence
G to A difference with canonical pSC101 vector sequence
p15A replication origin belongs to a different compatibility group from the common pMB1 (pBR or pUC) origin, and hence allows for co-existence with other pUC origin plasmids. When using this part, ligate it along with a proper antibiotic resistance gene and your gene of interest together to make a circular plasmid. Please do NOT include any other replication origin in your design.
Unlike pMB1 origin, which allows for chloramphenicol amplification, p15A replication origin is insensitive to chloramphenicol and remains in low copy number. This replication origin is different from the original one in that it contains several mutations, which increases the copy number to be no lower than pBR origin. In this way, both the compatibility and the convenience for use are accommodated.
OriT-RP4 (Origin of transfer for the RP4 plasmid)
Relaxation region of self-transmissible plasmid RP4. When this part is added to a plasmid, it is transferred by RP4 through conjugation.
This part is similar to J01003, which has no experience and is much longer. In the process of designing and testing this part, it will be compared to J01003.
This part is the standard BioBricks prefix.
Zymomonas mobilis glucose facilitator gene
NheI restriction enzyme site (XbaI, SpeI compatible overhang)
NheI recognition site. Digestion with NheI generates a compatible cohesive end to XbaI and SpeI.
This part is in BBb Format. It is flanked by BamHI and BglII 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] <!-- Add more about the biology of this part here
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
This part was constructed by PCR amplification of pSB3K3-P1010.
p15A origin of replication which is supposed to have low to medium copy number. It is also the origin of replication in pSB3* series plasmids.
The boundaries of this part are quite liberal to ensure that this part is a functional origin.
This part is taken from the RSF1010 derived plasmid pRL1383a. A description of this plasmid can be found at the NCBI database at accession number AF403426.
Plasmids containing oriV and the corresponding Rep proteins (from RSF1010) are autonomously replicated in a broad range of hosts. It has been reported that RSF1010 derived plasmids containing oriV and the Rep proteins are stably maintained in Pseudomonas (Bagdasarian 1981), Caulobacter (Umelo-Njaka et al. 2001), Erwinia, Serratia (Leemans 1987), and several cyanobacteria strains (Mermet-Bouvier 1993) including Synechocystis PCC6803 and PCC6714 and Synechococcus PCC7942 and PCC6301.
This part will be constructed with the origin of vegetative replication derived from an RSF1010 plasmid.
F plasmid backbone with BioBrick sites removed.
This is a (near) single copy origin derived from the F plasmid. The naturally occurring BioBricks sites were removed with an intent to preserve the amino acid and codon frequency where possible. Similar to the pSMART VC sequence from Lucigen http://www.lucigen.com/products/Cloning_Systems/CopyRight.html. It is intended to be combined with other vector components (antibiotic resistance marker, multiple cloning site) in order to form a BioBricks vector. Relevant information can be found at http://openwetware.org/wiki/BioBrick_Parts_for_Plasmid_Engineering.
Constitutive Promoter, High Transcription
Vikram Vijayan, Allen Hsu, Lawrence Fomundam
This part is to be constructed with the Rep proteins derived from an RSF1010 plasmid.
The GFP fusion brick was derived from the GFP BioBrick, [http://partsregistry.org/Part:BBa_E0040 BBa_E0040].
The first two nucleotides of the GFP start codon were removed to create this fusion brick.
slr2016 signal sequence from cyanobacterium Synechocystis; secretes protein
The ''slr2016'' signal sequence is located at the amino terminus of slr2016 polypeptides synthesized by ''Synechocystis'' sp. PCC 6803.
pSC101 temperature sensitive origin of replication
This part is derived from BBa_I50042. It contains a point mutation that changes Alanine to Valine in RepA protein.
BBa_I50052 is a pSC101 temperature sensitive origin of replication. BBa_I50052 is the temperature sensitive replication origin found in the pSB8* series of BioBrick vectors as well as the pSIM5 recombineering vector.
See design notes for Part:BBa_I50042.
This part is the replication origin from plasmid pBR322 which is supposed to have low to medium copy number.
Double terminator consisting of BBa_B0010 and BBa_B0012
Reverse of B0052, E.coli transcriptional terminator from the ribosomal RNA rrnC operon.
pSC101 heat sensitive origin of replication with deletion in par locus
A derivative of pSC101 origin of replication, this heat sensitive origin has the par locus deleted, therefore allowing a rapid loss of plasmid even under the permissive temperature.
The par locus was deleted through excision of BBa_K524000 by SspI and StuI, purification of desired fragments and then
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
This part is the standard BioBricks suffix.
atgcgtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtg aaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatgg tgttcaatgctttgcgagatacccagatcatatgaaacagcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttc aaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatg gaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaat tagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattac ctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatgg atgaactatacaaataataa ccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctc accttcgggtgggcctttctgcgtttata
The iGEM Team Panama 2011 have to design and assemble the ends of an expression platform in order to bring up by rational designing a fuctional test platform for the expression of our Team favorite BioBrick part BBa_K653000 A.K. The Biosurfactator. We choose to assembly two existing BioBrick parts from the shop, we mean The Registry. Those parts are: a GFP reporter BBa_E0040 and a double terminator BBa_B0015. We assemble them to bring up a new composite part that will serve as a well characterized ends for a expression platform for several coding sequences like our RhlAB_BBa_K653000
This GFP part was derived from the reporter device used by Elowitz's repressilator : GFPmut3* has a couple of mutations from wtGFP - S2R and S65G and S72A. Emerald and EGFP have S65T and multiple other mutations. The S2R mutation should have no effect. The Double terminator consisting of BBa_B0010 and BBa_B0012. This is the most commonly used terminator. It seems to be reliable. The assembling of this two parts was performed using the 3A Assembly Method.
Terminator to be placed upstream of multiple cloning site in new pSB series plasmids.
BBa_K864050 is a pSC101 origin of replication and is found in the pSB4*15 series of BioBrick vectors. This ori is an improvement of the ori found in all pSB4*5 plasmids, BBa_I50042. BBa_I50042 has been shown to have a higher copy number than expected.
This ori was created by cloning pSC101ts from pSIM5 and making a V56A mutation in repA.
PCR ca1011F/R on pG80ko (445 bp, EcoRI/SpeI) Sub into pSB1A2-I13522 (EcoRI/SpeI) Product is Bca1011 ---- ca1011F Forward Biobricking of R6K GGACTgaattcgcggccgcttctagagtgattcgcacgggcccatg ca1011R Reverse biobricking of R6K ccgctactagtaGCAGTTCAACCTGTTGATAG
R6K origin of replication requires a pir+ or pir116 strain for replication in the absence of a second origin. In most E. coli strains, this is a silent feature. {pSB1A2-Bca1011}
Transcriptional terminator consisting of a 64 bp stem-loop.
pSC101 origin of replication which is supposed to have low copy number. This was the origin used in Michael Elowitz's repressilator and in Bujard's pZS4Int-1 vector with mutations introduced to eliminate a SpeI site. It is also the origin of replication in pSB4* series plasmids. This part is the reverse copy of BBa_I50040.
difference from canonical pSC101 sequence in Genbank
green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212
GFP (mut3b) [note that this part does not have a barcode]
Terminator to be placed downstream of multiple cloning site in new pSB series plasmids. Similar to BBa_B0051.
identical to E. coli MG1655 between tonB and yciA
Topoisomerase I mediated cloning site (forward)
Insertion of this site will enable generation of a TOPO vector. The vector will have a T overhang and permit binding of topoisomerase I for direct cloning of PCR products. See http://openwetware.org/wiki/Topoisomerase_I_mediated_TA_cloning for a complete description of Topoisomerase I mediated cloning.
lac promoter/rbs/rep/oriV/lac promoter/rbs/aadA/oriT
This part was derived from RSF1010 and RP4.
This is an intermediate construction step for the construction of the broad-host-range plasmid derived from RSF1010. The coding region was inserted into pSB1A3.
I am having trouble inserting this part into the Base Vector so the construct will be tested while still in pSB1A3.
Minimal pUC19-derived high copy replication origin
High copy origin of replication derived from pSB1A3. Designed to omit antibiotic resistance markers and primer binding sites. Lacks mutations that render BBa_I50020 nonfunctional as a replication origin.
We had to leave several restriction sites in because point mutations that eliminated these sites were found to render the origin nonfunctional.
R6K minimal origin of replication. Only contains gamma origin and requires pir+ strain for replication. Copy number is dependent on amount of pir protein in the cell.
Topoisomerase I mediated cloning site (reverse)
Insertion of this site will enable generation of a TOPO vector. The vector will have a T overhang and permit binding of topoisomerase I for direct cloning of PCR products. See http://openwetware.org/wiki/Topoisomerase_I_mediated_TA_cloning for a complete description of Topoisomerase I mediated cloning. This site belongs 3' of the cloning site. It is the reverse complement of BBa_B0043.
pSC101ts temperature sensitive low copy origin of replication
pSC101ts is a temperature sensitive pSC101 repelication origin and propagates at 30???C but stop replicating at 42???C.
Reverse BioBrick primer annealing site (VR binding site)
This is the VR sequence to be used in construction of plasmids which include the VR primer site. It is the reverse complement of BBa_G00101.
3' UTR site of alcohol oxidase 1 gene (aox1)
The DNA sequence from plasmid pPIC9K of Invitrogen (http://products.invitrogen.com/ivgn/product/V17520?ICID==%3Dsearch-product) was used for primer design. But the genomic DNA of Pichia pastoris wildtype X33 was used for amplification via PCR.
3' UTR site of alcohol oxidase 1 gene (AOX1) from K. pastoris used as a recombination site.
nonfunctional pSC101 origin of replication
Conversion of the iGEM parts registry to SBOL2.1