载体基本信息载体基本信息

出品公司:	Invitrogen
载体名称:	pcDNA4/TO/Myc-His B
质粒类型:	哺乳动物表达载体；四环素诱导载体
高拷贝/低拷贝:	--
启动子:	CMV/TO
克隆方法:	多克隆位点，限制性内切酶
载体大小:	5155bp
5 测序引物及序列:	CMV forward 5’-CGCAAATGGGCGGTAGGCGTG-3’
3 测序引物及序列:	BGH Reverse: 5-TAGAAGGCACAGTCGAGG-3
载体标签:	His Tag (6x), Xpress Epitope Tag
载体抗性:	氨苄青霉素
筛选标记:	Zeocin
克隆菌株:	TOP10F´, DH5aF´, JM109, and INVaF´
宿主细胞（系）:	T-REx-293 R710-07； T-REx-HeLa R714-07； T-REx-U2OS R712-07； T-REx-Jurkat R722-07
备注:	pcDNA4/TO/myc-His B 载体是四环素诱导系统的应答载体，对应的调控载体是pcDNA6/TR； pcDNA4/TO/myc-His A,B,C的区别仅在多克隆位点处；
产品目录号:	V1030-20
稳定性:	稳表达或瞬表达
组成型:	诱导型
病毒/非病毒:	非病毒

载体质粒图谱和多克隆位点信息

pcDNA4-TO-myc-His B 载体图谱

载体简介

四环素诱导系统 The T-REx System yields higher levels of induced expression than any other regulated mammalian expression system. It utilizes the complete CMV promoter and adds control elements from the bacterial tetracycline resistance operon to effectively repress and derepress transcription from one of the strongest mammalian promoter sequences known (1,2). Specific Activation The T-REx System uses a repressor mechanism that blocks transcription from the powerful CMV promoter in the absence of tetracycline. Because the T-REx System elements do not use viral transactivators, you can achieve high-level expression from the complete CMV promoter without secondary, non-specific activation of host genes. The T-REx Mechanism The T-REx transcriptional control elements are illustrated in Figure 1. Two tetracycline operator sequences (TetO2) have been inserted between the TATA box of the CMV promoter and the transcriptional start site. The TetO2 sequence itself has no effect on expression. When the tetracycline repressor protein (TR) is present, it effectively binds the TetO2 sites and blocks transcription initiation. Tetracycline added to the culture medium binds to, and changes the conformation of, the TR protein. This change causes the TR protein to release the TetO2 sites, derepressing transcription from the CMV promoter. The result is high-level expression of the gene of interest (Figure 2). Expression levels can be modulated based on the tetracycline concentration and can be induced to levels that are achieved with constitutive CMV expression vectors. T-REx is a powerful inducible mammalian expression system that allows you to regulate expression from the complete human cytomegalovirus (CMV) enhancer-promoter. T-RExinducible expression vectors offer the following features: Complete CMV enhancer-promoter sequence containing two copies of the tetracycline operator TetO2 sequence for high-level regulated expression Zeocin or hygromycin resistance gene for effective selection of stable mammalian cell lines Large multiple cloning site to simplify cloning In addition, pcDNA4/TO/myc-His offers a c-myc epitope for rapid detection of the recombinant protein with an Anti-myc Antibody and a polyhistidine (6xHis) sequence for simple purification of the recombinant protein with nickel-chelating resin and detection with Anti- His(C-term) Antibody. The regulatory vector, pcDNA6/TR, is provided for high-level expression of the tetracycline repressor (TR) protein. This vector expresses the Blasticidin resistance gene for rapid selection of mammalian cell lines that stably express the TR protein.

载体序列

LOCUS       pcDNA4/TO/Myc-His B	5155 bp 	DNA	SYNDEFINITION  pcDNA4/TO/Myc-His BACCESSION   KEYWORDS    SOURCE        ORGANISM  other sequences; artificial sequences; vectors.FEATURES             Location/Qualifiers     source          1..5155                     /organism="pcDNA4/TO/Myc-His B"                     /mol_type="other DNA"     promoter        236..812                     /label="CMV_immearly_promoter"     misc_feature    315..602                     /label="CAG_enhancer"     misc_feature    769..789                     /label="CMV_fwd_primer"     promoter        770..893                     /label="CMV_promoter"     promoter        782..955                     /label="CMV2_promoter"     misc_feature    820..859                     /label="tetO"     misc_feature    869..893                     /label="LNCX_primer"     misc_feature    complement(1162..1179)                     /label="BGH_rev_primer"     terminator      1165..1392                     /label="bGH_PA_terminator"     rep_origin      1455..1761                     /label="f1_origin"     misc_feature    complement(1875..1895)                     /label="pBABE_3_primer"     misc_feature    complement(1881..2097)                     /label="SV40_enhancer"     promoter        1893..2162                     /label="SV40_promoter"     rep_origin      2061..2138                     /label="SV40_origin"     misc_feature    2123..2142                     /label="SV40pro_F_primer"     promoter        2256..2323                     /label="EM7_promoter"     gene            2324..2695                     /label="bleo"                     /gene="bleo"     misc_feature    2324..2698                     /label="sh_ble"     terminator      2831..2950                     /label="SV40_PA_terminator"     misc_feature    2919..2938                     /label="EBV_rev_primer"     promoter        complement(2994..3012)                     /label="M13_reverse_primer"     misc_feature    complement(3011..3033)                     /label="M13_pUC_rev_primer"     promoter        complement(3047..3076)                     /label="lac_promoter"     rep_origin      complement(3385..4004)                     /label="pBR322_origin"     gene            complement(4159..5019)                     /label="Ampicillin"                     /gene="Ampicillin"     CDS             complement(4159..5019)                     /label="ORF frame 2"                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA                     EIGASLIKHW*"     promoter        complement(5061..5089)                     /label="AmpR_promoter"                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA                     EIGASLIKHW*"ORIGIN    1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC TGCTCTGATG   61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG  121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC  181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT  241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA  301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC  361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC  421 ATTGACGTCA ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT  481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT  541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA  601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG  661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGAACC  721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG  781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CCCTATCAGT GATAGAGATC  841 TCCCTATCAG TGATAGAGAT CGTCGACGAG CTCGTTTAGT GAACCGTCAG ATCGCCTGGA  901 GACGCCATCC ACGCTGTTTT GACCTCCATA GAAGACACCG GGACCGATCC AGCCTCCGGA  961 CTCTAGCGTT TAAACTTAAG CTTGGTACCG AGCTCGGATC CACTAGTCCA GTGTGGTGGA 1021 ATTCTGCAGA TATCCAGCAC AGTGGCGGCC GCTCGAGTCT AGAGGGCCCG CGGTTCGAAC 1081 AAAAACTCAT CTCAGAAGAG GATCTGAATA TGCATACCGG TCATCATCAC CATCACCATT 1141 GAGTTTAAAC CCGCTGATCA GCCTCGACTG TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT 1201 GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC CTTTCCTAAT 1261 AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA TTCTATTCTG GGGGGTGGGG 1321 TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG CAGGCATGCT GGGGATGCGG 1381 TGGGCTCTAT GGCTTCTGAG GCGGAAAGAA CCAGCTGGGG CTCTAGGGGG TATCCCCACG 1441 CGCCCTGTAG CGGCGCATTA AGCGCGGCGG GTGTGGTGGT TACGCGCAGC GTGACCGCTA 1501 CACTTGCCAG CGCCCTAGCG CCCGCTCCTT TCGCTTTCTT CCCTTCCTTT CTCGCCACGT 1561 TCGCCGGCTT TCCCCGTCAA GCTCTAAATC GGGGCATCCC TTTAGGGTTC CGATTTAGTG 1621 CTTTACGGCA CCTCGACCCC AAAAAACTTG ATTAGGGTGA TGGTTCACGT AGTGGGCCAT 1681 CGCCCTGATA GACGGTTTTT CGCCCTTTGA CGTTGGAGTC CACGTTCTTT AATAGTGGAC 1741 TCTTGTTCCA AACTGGAACA ACACTCAACC CTATCTCGGT CTATTCTTTT GATTTATAAG 1801 GGATTTTGGG GATTTCGGCC TATTGGTTAA AAAATGAGCT GATTTAACAA AAATTTAACG 1861 CGAATTAATT CTGTGGAATG TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGG 1921 CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC 1981 CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCATAG 2041 TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC CAGTTCCGCC CATTCTCCGC 2101 CCCATGGCTG ACTAATTTTT TTTATTTATG CAGAGGCCGA GGCCGCCTCT GCCTCTGAGC 2161 TATTCCAGAA GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA AAGCTCCCGG 2221 GAGCTTGTAT ATCCATTTTC GGATCTGATC AGCACGTGTT GACAATTAAT CATCGGCATA 2281 GTATATCGGC ATAGTATAAT ACGACAAGGT GAGGAACTAA ACCATGGCCA AGTTGACCAG 2341 TGCCGTTCCG GTGCTCACCG CGCGCGACGT CGCCGGAGCG GTCGAGTTCT GGACCGACCG 2401 GCTCGGGTTC TCCCGGGACT TCGTGGAGGA CGACTTCGCC GGTGTGGTCC GGGACGACGT 2461 GACCCTGTTC ATCAGCGCGG TCCAGGACCA GGTGGTGCCG GACAACACCC TGGCCTGGGT 2521 GTGGGTGCGC GGCCTGGACG AGCTGTACGC CGAGTGGTCG GAGGTCGTGT CCACGAACTT 2581 CCGGGACGCC TCCGGGCCGG CCATGACCGA GATCGGCGAG CAGCCGTGGG GGCGGGAGTT 2641 CGCCCTGCGC GACCCGGCCG GCAACTGCGT GCACTTCGTG GCCGAGGAGC AGGACTGACA 2701 CGTGCTACGA GATTTCGATT CCACCGCCGC CTTCTATGAA AGGTTGGGCT TCGGAATCGT 2761 TTTCCGGGAC GCCGGCTGGA TGATCCTCCA GCGCGGGGAT CTCATGCTGG AGTTCTTCGC 2821 CCACCCCAAC TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA 2881 TTTCACAAAT AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA 2941 TGTATCTTAT CATGTCTGTA TACCGTCGAC CTCTAGCTAG AGCTTGGCGT AATCATGGTC 3001 ATAGCTGTTT CCTGTGTGAA ATTGTTATCC GCTCACAATT CCACACAACA TACGAGCCGG 3061 AAGCATAAAG TGTAAAGCCT GGGGTGCCTA ATGAGTGAGC TAACTCACAT TAATTGCGTT 3121 GCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC CAGCTGCATT AATGAATCGG 3181 CCAACGCGCG GGGAGAGGCG GTTTGCGTAT TGGGCGCTCT TCCGCTTCCT CGCTCACTGA 3241 CTCGCTGCGC TCGGTCGTTC GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT 3301 ACGGTTATCC ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA 3361 AAAGGCCAGG AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC 3421 TGACGAGCAT CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA 3481 AAGATACCAG GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC 3541 GCTTACCGGA TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCAATGCTC 3601 ACGCTGTAGG TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA 3661 ACCCCCCGTT CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC 3721 GGTAAGACAC GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG 3781 GTATGTAGGC GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG 3841 GACAGTATTT GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG 3901 CTCTTGATCC GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA 3961 GATTACGCGC AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA 4021 CGCTCAGTGG AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT 4081 CTTCACCTAG ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA 4141 GTAAACTTGG TCTGACAGTT ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG 4201 TCTATTTCGT TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA 4261 GGGCTTACCA TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC 4321 AGATTTATCA GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC 4381 TTTATCCGCC TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC 4441 AGTTAATAGT TTGCGCAACG TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC 4501 GTTTGGTATG GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC 4561 CATGTTGTGC AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT 4621 GGCCGCAGTG TTATCACTCA TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC 4681 ATCCGTAAGA TGCTTTTCTG TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG 4741 TATGCGGCGA CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG 4801 CAGAACTTTA AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT 4861 CTTACCGCTG TTGAGATCCA GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC 4921 ATCTTTTACT TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA 4981 AAAGGGAATA AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA 5041 TTGAAGCATT TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA 5101 AAATAAACAA ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTC

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