pcDNA5/FRT/TO-TOPO四環(huán)素調(diào)控誘導表達定向重組載體

描述：

pcDNA5/FRT/TO-TOPO載體是cDNA的表達與克隆載體；
 CMVTO啟動子受四環(huán)素調(diào)控；
 含有FRT位點（重組酶識別位點），與pOG44載體共轉(zhuǎn)染，利用重組酶進行定向重組；
 采用TOPO-TA技術(shù)，只用5分鐘即可將PCR片段連接到載體上去。

載體基本信息

出品公司:	Invitrogen
載體名稱:	pcDNA5/FRT/TO-TOPO
質(zhì)粒類型:	哺乳動物細胞表達載體；cDNA表達載體；定向重組載體；四環(huán)素調(diào)控載體
高拷貝/低拷貝:	高拷貝
克隆方法:	TOPO-TA
啟動子:	CMVTO
載體大小:	5155 bp
5' 測序引物及序列:	CMV Forward: 5’-CGCAAATGGGCGGTAGGCGTG-3’
3' 測序引物及序列:	BGH Reverse: 5’-TAGAAGGCACAGTCGAGG-3’
載體標簽:	無標簽
載體抗性:	氨芐青霉素
篩選標記:	潮霉素（Hygromycin）
克隆菌株:	TOP10, DH5α, JM109
宿主細胞（系）:	Invitrogen公司出品的Flp-In細胞系，293、CV-1、CHO等
備注:	pcDNA5/FRT/TO-TOPO載體是cDNA的表達與克隆載體；  CMVTO啟動子受四環(huán)素調(diào)控；  含有FRT位點（重組酶識別位點），與pOG44載體共轉(zhuǎn)染，利用重組酶進行定向重組；  采用TOPO-TA技術(shù)，只用5分鐘即可將PCR片段連接到載體上去。
產(chǎn)品目錄號:	K6510-20
穩(wěn)定性:	瞬表達 或 穩(wěn)表達
組成型/誘導型:	誘導型
病毒/非病毒:	非病毒

載體質(zhì)粒圖譜和多克隆位點信息

載體簡介

pcDNA5/FRT/TOTOPO 載體簡介pcDNA5/FRT/TO-TOPO is a 5.2 kb expression vector designed to facilitate rapid cloning and tetracycline-regulated expression of PCR products using the Flp-In T-REx System (Catalog no. K6500-01) available from Invitrogen. When cotransfected with the pOG44 Flp recombinase expression plasmid into a Flp-In T-REx mammalian host cell line, the pcDNA5/FRT/TO-TOPO vector containing the PCR product of interest is integrated in a Flp recombinase-dependent manner into the genome. Expression of the gene of interest can then be induced by addition of tetracycline to the culture medium.pcDNA5/FRT/TO, pcDNA5/FRT/TO-TOPO and pcDNA5/FRT/TO-E are designed for targeted Flp-In integration followed by tetracycline-regulated expression. Each vector contains a FRT site for efficient targeted integration and a hybrid CMV/TetO2 promoter for regulated expression. In addition, pcDNA5/FRT/TO-TOPO and pcDNA5/FRT/TO-E are adapted for TOPO-and Echo Cloning, respectively. TOPO Cloning enables rapid ligation of PCR products into pcDNA5/FRT/TO-TOPO in just five minutes on your bench top. Echo Cloning allows you to quickly shuttle your gene of interest into multiple expression vectors.The Flp-In T-REx 293 Cell Line is available to save time in expression experiments. This cell line contains pFRT/lacZeo and pcDNA6/TR stably integrated and is tested for its ability to regulate expression .pcDNA5/FRT/TO-TOPO 載體含有以下元件: A hybrid human cytomegalovirus (CMV)/TetO2 promoter for high-level, tetracycline-regulated expression of the gene of interest in a wide range of mammalian cells (Andersson et al., 1989; Boshart et al., 1985; Hillen and Berens, 1994; Hillen et al., 1983; Nelson et al., 1987) TOPO Cloning site for rapid and efficient cloning of Taq-amplified PCR products FLP Recombination Target (FRT) site for Flp recombinase-mediated integration of the vector into the Flp-In T-REx host cell line Hygromycin resistance gene for selection of stable cell lines (Gritz and Davies, 1983)The control plasmid, pcDNA5/FRT/TO/CAT, is included for use as a positive control for transfection and expression in the Flp-In T-REx host cell line of choice. For more information about the Flp-In T-REx System, the pOG44 plasmid, and generation of the Flp-In T-REx host cell line, refer to the Flp-In T-REx Core Kit manual. 雜交 CMV/TetO2 啟動子Expression of your gene of interest from pcDNA5/FRT/TO-TOPO is controlled by the strong human CMV immediate early enhancer/promoter (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987) into which 2 copies of the tet operator 2 (TetO2) sequence have been inserted in tandem. Insertion of these TetO2 sequences into the CMV promoter confers regulation by tetracycline to the promoter.The TetO2 sequences consist of 2 copies of the 19 nucleotide sequence, 5′-TCCCTATCAGTGATAGAGA-3′ separated by a 2 base pair spacer (Hillen and Berens, 1994; Hillen et al., 1983). Each 19 nucleotide TetO2 sequence serves as the binding site for 2 molecules of the Tet repressor. For more information about the mechanism of tetracycline regulation in the Flp-In T-REx System, refer to the Flp-In T-REx Core Kit manual.FRT 位點The pcDNA5/FRT/TO-TOPO vector contains a single FRT site immediately upstream of the hygromycin resistance gene for Flp recombinase-mediated integration and selection of the pcDNA5/FRT/TO-TOPO construct following cotransfection of the vector (with pOG44) into a Flp-In T-REx mammalian host cell line. The FRT site serves as both the recognition and cleavage site for the Flp recombinase and allows recombination to occur immediately adjacent to the hygromycin resistance gene. The Flp recombinase is expressed from the pOG44 plasmid. For more information about the FRT site and recombination, see below. For more information about pOG44, refer to the pOG44 manual or the Flp-In T-REx Core Kit manual.for Flp recombinase and has been well-characterized (Gronostajski and Sadowski, 1985; Jayaram, 1985; Sauer, 1994; Senecoff et al., 1985). The minimal FRT site consists of a 34 bp sequence containing two 13 bp imperfect inverted repeats separated by an 8 bp spacer that includes an Xba I restriction site. An additional 13 bp repeat is found in most FRT sites, but is not required for cleavage (Andrews et al., 1985). While Flp recombinase binds to all three of the 13 bp repeats, strand cleavage actually occurs at the boundaries of the 8 bp spacer region (Andrews et al., 1985; Senecoff et al., 1985).The hygromycin resistance gene in pcDNA5/FRT/TO-TOPO lacks a promoter and an ATG initiation codon; therefore, transfection of the pcDNA5/FRT/TO-TOPO plasmid alone into mammalian host cells will not confer hygromycin resistance to the cells. The SV40 promoter and ATG initiation codon required for expression of the hygromycin resistance gene are integrated into the genome (in the Flp-In T-REx host cell line) and are only brought into the correct proximity and frame with the hygromycin resistance gene through Flp recombinase-mediated integration of pcDNA5/FRT/TO-TOPO at the FRT site. Flp 重組酶介導的DNA重組In the Flp-InT-RExSystem, integration of your pcDNA5/FRT/TO-TOPO expression construct into the genome occurs via Flp recombinase-mediated intermolecular DNA recombination. The hallmarks of Flp-mediated recombination are listed below. Recombination occurs between specific FRT sites on the interacting DNA molecules Recombination is conservative and requires no DNA synthesis; the FRT sites are preserved following recombination and there is minimal opportunity for introduction of mutations at the recombination site Strand exchange requires only the small 34 bp minimal FRT siteTOPO克隆工作原理The plasmid vector, pcDNA5/FRT/TO-TOPO, is supplied linearized with: Single 3′ thymidine (T) overhangs for TA Cloning Topoisomerase covalently bound to the vector (this is referred to as “activated” vector) Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3′ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5′-CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3′ phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5′ hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994). TOPO Cloning exploits this reaction to efficiently clone PCR products .

pcDNA5/FRT/TO-TOPO載體表達實驗流程圖

載體序列

pcDNA5/FRT/TO-TOPO  5155bp

GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTT
AAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACA
ACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCG
ATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC
ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG
CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCC
ATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC
AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA
TGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC
AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG
CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATC
TCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCC
ACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAG
CTCGCCCTTAAGGGCGAGCTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATA
TCCAGCACAGTGGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCT
TCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCA
CTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGG
TGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGC
TCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCG
CATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGC
TCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGG
CTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTT
CACGTACCTAGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCTTGGCCAAAAA
GCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATG
CAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGG
TAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCC
GATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAG
GGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGG
ATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCA
ATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATG
GACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCG
AAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGC
GGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGG
CCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGC
CGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTT
CGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGT
ACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAA
ACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTACTACGAGATTTCGATTCCACCGCCGC
CTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGAT
CTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATA
GCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAA
TGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTT
CCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCT
GGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAA
CCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCT
TCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA
AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCA
AAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCAT
CACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCC
CTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC
TTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC
AAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTG
AGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG
GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTT
GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAA
CCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGA
AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTC
ATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAA
GTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTG
TCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCA
TCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACC
AGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTG
TTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGC
ATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTA
CATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTT
GGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGA
TGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT
CTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAA
ACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGT
GCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAA
ATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTA
TTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAA
ATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC