pcDNA5/FRT/TO基因定向重組載體四環(huán)素調(diào)控誘導(dǎo)表達(dá)載體

描述：

pcDNA5/FRT/TO載體是cDNA的表達(dá)與克隆載體；
 CMVTO啟動子受四環(huán)素調(diào)控；
 含有FRT位點(diǎn)（重組酶識別位點(diǎn)） ，與pOG44載體共轉(zhuǎn)染，利用重組酶進(jìn)行定向重組

載體基本信息

出品公司:	Invitrogen
載體名稱:	pcDNA5/FRT/TO
質(zhì)粒類型:	哺乳動物細(xì)胞表達(dá)載體；cDNA表達(dá)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:	限制性內(nèi)切酶，多克隆位點(diǎn)
啟動子:	CMVTO（CMV/TetO2）
載體大小:	5137 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(biāo)簽:	無標(biāo)簽
載體抗性:	氨芐青霉素
篩選標(biāo)記:	潮霉素（Hygromycin）
克隆菌株:	TOP10, DH5α, JM109
宿主細(xì)胞（系）:	Invitrogen公司出品的Flp-In細(xì)胞系，293、CV-1、CHO等
備注:	pcDNA5/FRT/TO載體是cDNA的表達(dá)與克隆載體；  CMVTO啟動子受四環(huán)素調(diào)控；  含有FRT位點(diǎn)（重組酶識別位點(diǎn)） ，與pOG44載體共轉(zhuǎn)染，利用重組酶進(jìn)行定向重組。
產(chǎn)品目錄號:	V6520-20
穩(wěn)定性:	瞬表達(dá) 或 穩(wěn)表達(dá)
組成型/誘導(dǎo)型:	誘導(dǎo)型
病毒/非病毒:	非病毒

載體質(zhì)粒圖譜和多克隆位點(diǎn)信息

載體簡介

簡介pcDNAFRTTO vector is a 5.1 kb inducible expression vector designed for use with the Flp-In T-REx System. The control plasmid, pcDNA5FRTTOCAT, is also included for use as a positive control for transfection and expression in your Flp-In T-REx host cell line of choice.pcDNA5/FRT/TO is a 5.1 kb inducible expression vector designed for use with the Flp-In T-REx System (Cat. 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 vector containing the gene of interest is integrated in a Flp recombinase-dependent manner into the genome. Expression of the gene of interest may be induced by the addition of tetracycline to the culture medium. pcDNA5/FRT/TO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) Multiple cloning site with 10 unique restriction sites to facilitate cloning the gene of interest 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 is controlled by the strong 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.關(guān)于pcDNA5/FRT/TO需要注意的事項(xiàng)The pcDNA5/FRT/TO 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 plasmid following cotransfection of the vector (with pOG44) into Flp-In T-REx mammalian host cells. 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.The hygromycin resistance gene in pcDNA5/FRT/TO lacks a promoter and an ATG initiation codon; therefore, transfection of the pcDNA5/FRT/TO 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 at the FRT site.Flp 重組酶介導(dǎo)的DNA重組In the Flp-In T-REx System, integration of your pcDNA5/FRT/TO inducible 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 (see below) 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 siteFRT 位點(diǎn)The FRT site, originally isolated from Saccharomyces cerevisiae, serves as a binding site 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

pcDNA5/FRT/TO 載體使用流程如下：

載體序列

pcDNA5/FRT/TO  5137 bp

GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTT
AAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACA
ACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCG
ATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTC
ATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCG
CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCC
ATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCC
AAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTA
TGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGC
AGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAA
TGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACG
CAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCCCTATCAGTGATAGAGATC
TCCCTATCAGTGATAGAGATCGTCGACGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCC
ACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGCGTTTAAACTTAAG
CTTGGTACCGAGCTCGGATCCACTAGTCCAGTGTGGTGGAATTCTGCAGATATCCAGCACAGTGGCGGCC
GCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCT
GTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA
ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAG
CAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCG
GAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTG
TGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCC
TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGA
TTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTACCTAGAAGTTCC
TATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCCTTGGCCAAAAAGCCTGAACTCACCGCGAC
GTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAA
GAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATG
GTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGA
CATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGAC
CTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCG
ATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGA
TTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCG
TCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGC
ACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGA
GGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATG
GAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATA
TGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGC
GCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGA
AGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTC
GTCCGAGGGCAAAGGAATAGCACGTACTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGG
CTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTC
GCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAA
ATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTG
TATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTAT
CCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGA
GCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA
TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACT
GACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTAT
CCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAA
AAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCA
AGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGC
GCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCT
TTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCAC
GAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGAC
ACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTAC
AGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTG
AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTG
GTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTC
TACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG
ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTT
GGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCAT
AGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCA
ATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCG
AGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGT
AAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCG
TCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGT
GCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACT
CATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGT
GAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATAC
GGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAA
ACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCA
GCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAA
TAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGG
TTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA
TTTCCCCGAAAAGTGCCACCTGACGTC