Target plasmid for three antibiotic assembly. Chloramphenicol resistant. Contains an insert having a constitutive promoter and the ccdB coding region. ccdB protein is lethal in normal cloning strains. Grown in the special strain DB3.1, which is ccdB resistant.
F plasmid with OriT and TraJ knocked out by lambda-red
Target plasmid for three antibiotic assembly. The ccdB insert prevents growth of the unmodified plasmid in normal cloning strains, allowing selection of only correctly assembled recombinants.
The rhlR coding sequence on vector J61002
This plasmid contains the ColE1 origin of replication, kanamycin resistance marker, and tet0-1 promoter.
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
This plasmid has the pSC101* origin of replication, kanamycin resistance marker, and the lac/ara promoter.
This plasmid has the ColE1 origin of replication, amp resistance marker, lac0-1 promoter, and multiple cloning sites.
Plasmid with p15A origin of replication, Chloramphenicol resistance, and tet-0 promoter
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.
oriV origin which requires the presence of the RK2-encoded TrfA replication protein (resident on the genome of a host-strain) to be functional. Vector copy number will be 20-50 if this replication origin is active. For this origin to be functional, the strain background must contain the trfA gene. Generally the appropriate strain has a copy-up TrfA mutant protein controlled by the ParaBAD promoter. Thus, L-arabinose can be added to the media to induce expression of the TrfA protein and enable replication at the oriV origin. The origin will not be functional in host strains without this protein. Generally, this origin is used as a secondary, inducible copy number origin in F plasmid based vectors. It allows conditional amplication of the vector copy number when in the appropriate host strain. Similar to part of 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, a single copy origin like BBa_I50000) in order to form a BioBricks vector. Relevant information can be found at http://openwetware.org/wiki/BioBrick_Parts_for_Plasmid_Engineering.
Plasmid with ColE1 origin of replication, amp resistance marker, and A1lac0 promoter with multiple cloning sites
This is a complete plasmid with the pSC101 origin of replication, spectinomycin resistance marker, and lac/ara promoter
Target plasmid for three antibiotic assembly. Tetracycline resistant. Contains an insert with a constitutive promoter and the ccdB coding region. The ccdB gene expression is lethal in normal cloning strains. The plasmid must be replicated in the special ccdB resistant strain DB3.1.
Plasmid for Chromosomal Integration in Yeast at Ura3
This part is designed to allow simple chromosomal integration of BioBrick plasmids. It is based off of the bacterial pSB1AT3 backbone allowing for normal DNA manipulation to occur in bacteria. However the BioBrick cut sites are flanked by homologous regions to the Ura3 locus in S. cerevisiae. To transform S. cerevisiae the plasmid is cut with BbsI creating two non-sticky ends. The linear fragment then contains Ura3 homologous sequences at its ends that can be used to chromosomally integrate the BioBrick plasmid into the yeast genome. Transformants can then be selected for using Ura- medium.
This part was derived from plasmid pSB1AT3. Two successive rounds of PCR inserted a BbsI cut site and 40 bp homologous region in before the prefix and after the suffix. The following Primers were used: Forward 1 GCACAGAACAATAACCTGCTGGAAACGAAGATAAATCgaagacGATTACTTCGCGTTATGCAGGC Reverse 1 GCATCTTCTCAAATATGCTTCCCAGCCTGCTTATCcttctgAAATTCTGCCTCGTGATACGCC Forward 2 tactagtagcggccgctgcagTCTTAACCCAACTGCACAGAACAATAACCTGCTGGAAACG Reverse 2 ctctagaagcggccgcgaattcTTAGTATTGCTGGCCGCATCTTCTCAAATATGCTTCCCAGCC
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.
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.
C to G mutation (eliminates XbaI site, noncoding region)
sequence derived from F plasmid in GenBank Accession Number AP001918
ori2 (oriS) single copy replication origin
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. "