Digest pBca9145-I716206 (BamHI/AlwnI, 1694+553bp, Large) Digest pBca9145-Bca1114 (BglII/AlwnI, 1519+593bp, Small) Product is pBca9145-I716208.
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 9 AA linker as spacer (BBa_K1680001). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 15 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 15 AA linker as spacer (BBa_K1680003). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
Cre recombinase gene, with a spacer for the rbs prepended. ---- 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]
PCR mv025/mv002 on pBca9145-Bca1010 (1067bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112115 ----------------------------------------------- mv025 Forward Biobricking of a~Cre! ccataGAATTCatgAGATCTttaaatgtccaatttactgaccgtacacc mv002 Reverse Biobricking of <Cre! cgttaGGATCCttaatcgccatcttccagc
PCR sil06 and G00101 on pBca9145-Bca1117 (PCR product is 1147bp, BglII/XhoI 1047 bp) Sub into pBca9145-Bca1089 (BglII/XhoI, 2063+696, L) Product is pBca9145-I716213 (TTG start) sil06 Forward BglII Cre with TTG start ctagcAGATCTttgtccaatttactgaccgtac G00101 Reverse Sequencing of pSB1A* plasmids attaccgcctttgagtgagc
PCR sil06 and G00101 on pBca9145-Bca1117 (PCR product is 1147bp, BglII/XhoI 1047 bp) Sub into pBca9145-Bca1089 (BglII/XhoI, 2063+696, L) Product is pBca9145-I716213 (TTG start)
This part encodes a reporter cassette for the Cre Recombinase (e.g parts XYZ) in Yeast. An active Cre protein will switch expression from RFP to NanoLuc luciferase.
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with intermediate 6 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 12 AA linker as spacer (BBa_K1680002). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
This part is the SNAP UNIT of cyclebow system including an sv40 nuclear location sequence (NLS), a covalent labeling of fusion proteins named SNAP-tag?? and an sv40 terminator flanked by homodromous recognition site of recombinase CRE named loxP. The whole part can be cut off by CRE. You may use this part to: 1) Localize proteins in vivo with BLOCK and dye from NEB company; 2) Dye after blocking, you are able to see whether an event have an influence on the localization and expression of the interested protein; 3) Fast cut off this part by transient transfecting recombinase gene into nucleus.
The SNAP-tag?? was brought from NEB company. NLS and sv40 terminator were got from commercial plasmid: pentry nls GFP/pcdna3.1.
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 12 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
The antibody for the c-myc transcription factor. This protein binds to Enhancer Box sequences and recruits histone acetyltransferases, serving as a traditional transcription factor and a regulator of global chromatin structure.
The coding region of the gene was isolated via PCR. Biobrick prefix and suffix were added during amplification, and ligated into Ampicillin resistant plasmids.
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 9 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
This part is built in Biobrick version 2.0. In this format, parts are flanked by BglII and BamHI restriction sites. --------------------------------------------- PCR mv003/mv004 on pBca9145-Bca1010 (1060bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112101 ----------------------------------------------- mv003 Forward Biobricking of Cre> ccataGAATTCatgAGATCTatgtccaatttactgaccgtac mv004 Reverse Biobricking of Cre> cgttaGGATCCatcgccatcttccagcaggcg <//pre>
PCR mv003/mv004 on pBca9145-Bca1010 (1060bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112101
This part is built in Biobrick version 2.0. In this format, parts are flanked by BglII and BamHI restriction sites. PCR mv001/mv002 on pBca9145-Bca1010 (1060bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112100 ----------------------------------------------- mv001 Forward Biobricking of <Cre! ccataGAATTCatgAGATCTtccaatttactgaccgtacacc mv002 Reverse Biobricking of <Cre! cgttaGGATCCttaatcgccatcttccagc
PCR mv001/mv002 on pBca9145-Bca1010 (1060bp, EcoRI/BamHI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112100
Recombination Device composed by Cre-lox71
This device was created to express the Cre recombinase enzyme and a target gene under the control of the same promoter (promoter not included in this part). The target gene needs to be amplified by PCR, using primers that will insert a loxP and a lox66 sites flanking the sequence, in order to integrate it in the lox71 recombination site. For this part the lox71 is located downstream the Cre recombinase gene. An alternative construction is submitted in the part BBa_K886001. Once this device is located under the control of the desired promoter, it will express the Cre recombinase that recognizes the lox sites and inserts the target gene under the control of the same promoter. The Cre expression levels are critical for the recombination, we created two different devices: BBa_K886002 (Cre-Lox71) and BBa_K886001 (Lox71-Cre) for testing if inserting the target gene either upstream or downstream the Cre gene will affect the expression efficiency of the target gene
We used the lox71 submitted by our group BBa_K886000, which corrected the parts BBa_I718017 and BBa_K537020. Those parts are wrong when compared with the published sequence at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/
We synthesized the lox71 site, then we assembled it downstream the Cre recombinase gene(BBa_J61047).
This part is built in Biobrick version 2.0. In this format, parts are flanked by BglII and BamHI restriction sites. ---------------------------------------- PCR mv001/mv004 on pBca9145-Bca1010 (1057bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112102 ----------------------------------------------- mv001 Forward Biobricking of <Cre! ccataGAATTCatgAGATCTtccaatttactgaccgtacacc mv004 Reverse Biobricking of Cre> cgttaGGATCCatcgccatcttccagcaggcg
PCR mv001/mv004 on pBca9145-Bca1010 (1057bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112102
The particularity of lox71 is that it has an altered sequence at the end of it's right arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm.
Based on the report made by the igem2010 UT-Tokyo team and some papers, the lox71 (BBa_I718017) from the registry was wrong, it had a cg instead a gc in its center sequence that is between the arms. Apparently, this part was corrected by the iGEM11_WITS_CSIR_SA team (BBa_K537020), but the sequence from this group had the same error. The corrected part from iGEM11_Tokyo_Tech (BBa_K649205) was right but no DNA was available in the registry. So we decided to synthesized it and test it, using the proper sequence described by http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/, and we submitted the correct DNA to the Registry.
Protein coding region for the DNA recombinase Cre, codon optimized for yeast. The Cre recombinase recognizes two loxP sites (Part: BBa_K1680005) and depending on their orientation cuts out or reverses the DNA sequence between them.
the Cre has a GTG start to weaken it's activity
Lox71 is a site specific recombination cassette. It belongs to the loxP family frequently used in genetics, particularly in mouse genetics. lox site recombination is catalysed by a Site specific recombinase, Cre. lox sequences are composed of an 8 bp Core sequence surrounded by two Arms. The particularity of lox66 is that it has an altered sequence at the end of it's left arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm. As a consequence, after a recombination between a lox71 and a lox66 (altered right arm sequence), one of the two resulting generated lox sites has very low recombination potential as it inherited both mutated arms. Use of lox71 & lox66 sites is potentially interesting when the recombination reaction must be "irreversible".
No modifaication was made on lox71 sequence
This part was generated in the form of a forward & a reverse primer. After annealing these primers EcoRI & PstI compatible cohesive ends at the 5' & 3' ends of the dsDNA were generated. Next, the dsDNA was subcloned in a pSB1A2 open plasmid (digested with EcoRI & PstI) You can follow the construction process by following the links available in the Paris iGEM 2007 wiki: http://parts.mit.edu/igem07/index.php/Paris "freezer" section plasmids table. A links sends you to the corresponding notebook date when the ligation reaction was performed
lox66 is a site specific recombination cassette. It belongs to the loxP family frequently used in genetics, particularily in mouse genetics. lox site recombination is catalysed by a Site specific recombinase, Cre. lox sequences are composed of an 8 bp Core sequence surrounded by two Arms. The particularity of lox66 is that it has an altered sequence at the end of it's left arm compared to loxP. This sequence variation reduces affinity of the Cre recombinase for the arm. As a consequence, after a recombination between a lox66 and a lox71 (altered right arm sequence), one of the two resulting generated lox sites has very low recombination potential as it inherited both mutated arms. Use of lox66 & lox71 sites is potentially interresting when the recombination reaction must be "irreversible".
No modidification was made on the lox66 sequence
Recombination Device composed by lox71-Cre recombinase
This device was created to express the Cre recombinase enzyme and a target gene under the control of the same promoter (promoter not included in this part). The target gene needs to be amplified by PCR using primers that will insert a loxP and a lox66 sites flanking the sequence in order to integrate it in the lox71 recombination site located upstream the Cre recombinase gene. Once this device is located under the control of the desired promoter, it will express the Cre recombinase that recognizes the lox sites and inserts the target gene under the control of the same promoter. The Cre expression levels are critical for the recombination, we created two different devices: A (Cre-Lox71) and B (Lox71-Cre) for testing if inserting the target gene either upstream or downstream the Cre gene will affect the expression efficiency of the target gene.
We used the lox71 submitted by our group, which correct the part BBa_I718017 and BBa_K537020 when compared with the published sequence at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137435/
We synthesized the lox71 site and then we assembled it with the Cre recombinase part BBa_J61047
weakened version of Cre (has a GTG start codon) because we want a lower copy
PCR sil05 and G00101 on pBca9145-Bca1117 (PCR product is 1147bp, BglII/XhoI 1047 bp) Sub into pBca9145-Bca1089 (BglII/XhoI, 2063+696, L) Product is pBca9145-I716212 (GTG start) sil05 Forward BglII Cre with GTG start ctagcAGATCTgtgtccaatttactgaccgtac G00101 Reverse Sequencing of pSB1A* plasmids attaccgcctttgagtgagc
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 6 AA linker as spacer (BBa_K1680000). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
This part contains the WT loxP site, which is recognised by Cre recombinase (Part BBa_K1680007), in forward orientation.
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
Protein coding region for a mutant (Deletion of AA1-19) of DNA recombinase Cre, codon optimized for yeast.