This is the protein genertors of Rci site-specific recombinase BBa_K361000, which is being linked with a promotor and a ribosome binding site in front of it, a double terminator after it.
This is the optimal system for Rci site-specific recombinase production.
The promotor is come from lacI regulated one: BBa_R0010 The ribosome binding site is come from the defines RBS efficiency equal to one : BBa_B0034 The terminator is come from a doble terminator with bidirectional termination: BBa_B0014
This part produces the Hin protein with an LVA degredation tag. We are currently looking into this as a way to ensure no Hin is floating around once we turn off the Hin promoter.
Salmonella typhimurium and the the Hin part without LVA.
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
excisionase from E. coli phage P22 (removes prophage from host genome)
{rbs.int!} The integrase gene with native rbs site and stop codon, BBb format
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]
recT recombination gene from Spiroplasma citri
recET recombination gene from Spiroplasma citri, probably catalyzing the homologous recombination of DNA fragments similar to the Lambda Bet gene (Lambda RED recombination). This gene is from S. citri, a close relative of Me. florum, and has very similar codon usage.
Eliminate GATC site by silent mutation to GACC (asp -> asp). Note very low GC content at the 5' end. The primers are: * gtttcttcgaattcgcggccgcttctagatgaataataaaattgaattaatattaaataattc,recT-F * gtttcttctactagtagcggccgctgcagttattaatcagaaaacatttcattaacagc,recT-R * gaaatgcaagaagcctatcaacatgaccaagcaataattattaataacagc,recT-mut-F * gctgttattaataattattgcttggtcatgttgataggcttcttgcatttc,recT-mut-R
Genbank entry CAK99381 protein, nucleotide coding region. Mutations of the single GATC site to avoid restriction by the Sau3AI restriction system in Me. florum. PCR from S. citri DNA, kind gift of Prof. J. Renaudin.
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)
Mycobacterium Phage Bxb1 gp47, DNA excisionase
This part is the protein coding sequence which encodes DNA integrase of Mycobacterium phage Bxb1. The Bxb1 integrase is a DNA recombinase, more precisely a member of serine integrase family. It recognizes specific sequences, called attB and attP, and then integrate, invert, or excise depend on orientations of recognition sequences. We used this integrase to invert specific sequence contained in plasmid. This protein is well expressed in E.coli(strain MG1655). When it inverts DNA, the attB and attP sequences are changed into attL and attR, as common DNA recombinases do. Another protein called Bxb1 excisionase interacts with integrase and that complex flips back inverted DNA into original sequence regenerating attB and attP sequences.
Internal restriction endonuclease site is changed(two PstI sites, one XbaI sites) without changing amino acid sequence.
This part is derived by Mycobacterium phage Bxb1. And synthesized by Bioneer. http://www.ncbi.nlm.nih.gov/protein/NP_075302.1
The VIKA UNIT: recombinase VIKA flanked by Vox
This part is the VIKA UNIT of cyclebow system including an sv40 nuclear location sequence (NLS), a recombinase gene named VIKA and an sv40 terminator flanked by homodromous recognition site of recombinase VIKA named Vox. The whole part can be cut off by VIKA. This part is the improvement of the BBa_K1641003，which is a plain VIKA. You may use this part to: 1) Test the cut-off efficiency of recombinase VIKA by qRT-PCR; 2) Fast cut off this part by transient transfecting recombinase gene into nucleus; 3) Do lineage tracking.
The recombinase gene VIKA and its RTS Vox were got through denovo synthesis. NLS and sv40 terminator were got from commercial plasmid: pentry nls GFP/pcdn3.1.
excisionase from E. coli phage lambda (removes prophage from host genome)
Lambda excisionase coding region with AAV degradation tag and two stop codons
{int!} The integrase gene with start and stop codon, BBb format
{rbs.ihfB!} The ihf beta gene with native rbs and stop codon, BBb format
{rbs.ihfA!} The ihf alpha gene with native rbs and stop codon, BBb format
{int>} The integrase gene without a stop codon, BBb format
Mycobacterium Phage Bxb1 gp35, DNA integrase
lacA-homology sequence(for RED recombination system)
Saccharomyces cerevisiae 2 micron circle plasmid
{pAH57 promoter}{rbs.xis.int!} inserted into K112995 plasmid
This part was inserted into the K112995 plasmid between ccdB and the attR2 site. 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]
{ihfA>} The ihf alpha gene without a stop codon, BBb format
{a~int!} The integrase gene with rbs ready to be attached with stop codon, BBb format
{a~ihfA!} The ihf alpha gene ready to have rbs attached with stop codon, BBb format
component of RuvABC resolvasome, regulatory subunit
PCR using primers with biobrick prefix and surfix, and then cloned into pSB1AC3.
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
{< int!} The integrase gene without a start codon, BBb format
{< ihfA >} The ihf alpha gene without start and stop codons, BBb format
Intermediate of the oriTr (RP4 origin of transfer) and R6K (conditional origin of replication) pSB1A2-Bca9009
{< ihfB >} The ihf beta gene without start or stop codons, BBb format
{< ihfA!} The ihf alpha gene without a start codon, BBb format
This part is the SNAP-VIKA UNIT of cyclebow including a SNAP UNIT and a VIKA UNIT. The SNAP UNIT, which can be cut off by CRE, includes 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 VIKA UNIT, which can be cut off by VIKA, includes an sv40 nuclear location sequence (NLS), a recombinase gene named VIKA and an sv40 terminator flanked by homodromous recognition site of recombinase VIKA named Vox. You may use this part to: 1) Test the cut-off efficiency of recombinase CRE and VIKA by qRT-PCR; 2) Estimate the time needed for genome repair because VIKA can only be expressed after the cut off of SNAP UNIT and genome repair; 3) Fast cut off this part by transient transfecting recombinase gene into nucleus; 4) When there is no CRE, you can use this part as the SNAP UNIT, which can help localize proteins in vivo with BLOCK and dye from NEB company.
The SNAP-tag?? was brought from NEB company. NLS and sv40 terminator were got from commercial plasmid: pentry nls GFP/pcdna3.1. The recombinase gene VIKA and its RTS Vox were got through denovo synthesis.
{ihfB>} The ihf beta gene without a stop codon, BBb format
ATP-dependent DNA helicase, component of RuvABC resolvasome
This part will prouce a 384-amino acid Rci site-specific recombinase used in shufflon system, which is a site-specific DNA recombination between two specific repeat sites as recognition. After this recombinase is being expressed, it can alter the DNA sequence being inserted randomly with the specific repeat sites originally to become an unknown sequence.
The gene is come from muliple inversion systems in R64 plasmid and there is no further modification. Reports have shown that some modification of the gene will lead to deletion some parts of DNA sequence during recombination process.
It belongs to integrase family of site-specific recombinase, and it is extracted from muliple inversion systems in R64 plasmid, found in genomic sequence code:AP005147.1
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
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
3' UTR site of alcohol oxidase 1 gene (aox1)
3' UTR site of alcohol oxidase 1 gene (AOX1) from K. pastoris used as a recombination site.
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.
{< int >} The integrase gene without start and stop codons, BBb format
component of RuvABC resolvasome, endonuclease
DNA-invertase Hin from Salmonella typhimurium
Generates the Salmonella typhimurium Hin protein. This protein is used to invert DNA that is flanked by Hix siteds.
{a~ihfB!} The ihf beta gene ready to have rbs attached and stop codon, BBb format
{ihfB!} The ihf beta gene with start and stop codons, BBb format
{< ihfB!} The ihf beta gene without a start codon, BBb format