pcDNA4/TO/Myc-His C

pcDNA4/TO/Myc-His C

载体基本信息

出品公司:	Ybscience
载体名称:	pcDNA4/TO/myc-His C
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:	高拷贝
启动子:	CMV/TO
克隆方法:	多克隆位点，限制性内切酶
载体大小:	5147 bp
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´
表达细胞（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；
稳定性:	瞬表达 或 稳表达
组成型:	诱导型（四环素）
病毒/非病毒:	非病毒

 

载体质粒图谱和多克隆位点信息

载体简介

 

T-REx四环素诱导系统
A Tetracycline-Regulated Expression System without Viral Transactivators
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 C	5147 bp 	DNA	SYN
DEFINITION  pcDNA4/TO/Myc-His C
ACCESSION   
KEYWORDS    
SOURCE      
  ORGANISM  other sequences; artificial sequences; vectors.
FEATURES             Location/Qualifiers
     source          1..5147
                     /organism="pcDNA4/TO/Myc-His C"
                     /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(1154..1171)
                     /label="BGH_rev_primer"
     terminator      1157..1384
                     /label="bGH_PA_terminator"
     rep_origin      1447..1753
                     /label="f1_origin"
     misc_feature    complement(1867..1887)
                     /label="pBABE_3_primer"
     misc_feature    complement(1873..2089)
                     /label="SV40_enhancer"
     promoter        1885..2154
                     /label="SV40_promoter"
     rep_origin      2053..2130
                     /label="SV40_origin"
     misc_feature    2115..2134
                     /label="SV40pro_F_primer"
     promoter        2248..2315
                     /label="EM7_promoter"
     misc_feature    2316..2690
                     /label="sh_ble"
     gene            2316..2687
                     /label="bleo"
                     /gene="bleo"
     terminator      2823..2942
                     /label="SV40_PA_terminator"
     misc_feature    2911..2930
                     /label="EBV_rev_primer"
     promoter        complement(2986..3004)
                     /label="M13_reverse_primer"
     misc_feature    complement(3003..3025)
                     /label="M13_pUC_rev_primer"
     promoter        complement(3039..3068)
                     /label="lac_promoter"
     rep_origin      complement(3377..3996)
                     /label="pBR322_origin"
     gene            complement(4151..5011)
                     /label="Ampicillin"
                     /gene="Ampicillin"
     CDS             complement(4151..5011)
                     /label="ORF frame 2"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW*"
     promoter        complement(5053..5081)
                     /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 GCTCGAGGTC ACCCATTCGA ACAAAAACTC
 1081 ATCTCAGAAG AGGATCTGAA TATGCATACC GGTCATCATC ACCATCACCA TTGAGTTTAA
 1141 ACCCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CATCTGTTGT TTGCCCCTCC
 1201 CCCGTGCCTT CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA ATAAAATGAG
 1261 GAAATTGCAT CGCATTGTCT GAGTAGGTGT CATTCTATTC TGGGGGGTGG GGTGGGGCAG
 1321 GACAGCAAGG GGGAGGATTG GGAAGACAAT AGCAGGCATG CTGGGGATGC GGTGGGCTCT
 1381 ATGGCTTCTG AGGCGGAAAG AACCAGCTGG GGCTCTAGGG GGTATCCCCA CGCGCCCTGT
 1441 AGCGGCGCAT TAAGCGCGGC GGGTGTGGTG GTTACGCGCA GCGTGACCGC TACACTTGCC
 1501 AGCGCCCTAG CGCCCGCTCC TTTCGCTTTC TTCCCTTCCT TTCTCGCCAC GTTCGCCGGC
 1561 TTTCCCCGTC AAGCTCTAAA TCGGGGCATC CCTTTAGGGT TCCGATTTAG TGCTTTACGG
 1621 CACCTCGACC CCAAAAAACT TGATTAGGGT GATGGTTCAC GTAGTGGGCC ATCGCCCTGA
 1681 TAGACGGTTT TTCGCCCTTT GACGTTGGAG TCCACGTTCT TTAATAGTGG ACTCTTGTTC
 1741 CAAACTGGAA CAACACTCAA CCCTATCTCG GTCTATTCTT TTGATTTATA AGGGATTTTG
 1801 GGGATTTCGG CCTATTGGTT AAAAAATGAG CTGATTTAAC AAAAATTTAA CGCGAATTAA
 1861 TTCTGTGGAA TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GGCAGGCAGA
 1921 AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCAGGT GTGGAAAGTC CCCAGGCTCC
 1981 CCAGCAGGCA GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAT AGTCCCGCCC
 2041 CTAACTCCGC CCATCCCGCC CCTAACTCCG CCCAGTTCCG CCCATTCTCC GCCCCATGGC
 2101 TGACTAATTT TTTTTATTTA TGCAGAGGCC GAGGCCGCCT CTGCCTCTGA GCTATTCCAG
 2161 AAGTAGTGAG GAGGCTTTTT TGGAGGCCTA GGCTTTTGCA AAAAGCTCCC GGGAGCTTGT
 2221 ATATCCATTT TCGGATCTGA TCAGCACGTG TTGACAATTA ATCATCGGCA TAGTATATCG
 2281 GCATAGTATA ATACGACAAG GTGAGGAACT AAACCATGGC CAAGTTGACC AGTGCCGTTC
 2341 CGGTGCTCAC CGCGCGCGAC GTCGCCGGAG CGGTCGAGTT CTGGACCGAC CGGCTCGGGT
 2401 TCTCCCGGGA CTTCGTGGAG GACGACTTCG CCGGTGTGGT CCGGGACGAC GTGACCCTGT
 2461 TCATCAGCGC GGTCCAGGAC CAGGTGGTGC CGGACAACAC CCTGGCCTGG GTGTGGGTGC
 2521 GCGGCCTGGA CGAGCTGTAC GCCGAGTGGT CGGAGGTCGT GTCCACGAAC TTCCGGGACG
 2581 CCTCCGGGCC GGCCATGACC GAGATCGGCG AGCAGCCGTG GGGGCGGGAG TTCGCCCTGC
 2641 GCGACCCGGC CGGCAACTGC GTGCACTTCG TGGCCGAGGA GCAGGACTGA CACGTGCTAC
 2701 GAGATTTCGA TTCCACCGCC GCCTTCTATG AAAGGTTGGG CTTCGGAATC GTTTTCCGGG
 2761 ACGCCGGCTG GATGATCCTC CAGCGCGGGG ATCTCATGCT GGAGTTCTTC GCCCACCCCA
 2821 ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA
 2881 ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT
 2941 ATCATGTCTG TATACCGTCG ACCTCTAGCT AGAGCTTGGC GTAATCATGG TCATAGCTGT
 3001 TTCCTGTGTG AAATTGTTAT CCGCTCACAA TTCCACACAA CATACGAGCC GGAAGCATAA
 3061 AGTGTAAAGC CTGGGGTGCC TAATGAGTGA GCTAACTCAC ATTAATTGCG TTGCGCTCAC
 3121 TGCCCGCTTT CCAGTCGGGA AACCTGTCGT GCCAGCTGCA TTAATGAATC GGCCAACGCG
 3181 CGGGGAGAGG CGGTTTGCGT ATTGGGCGCT CTTCCGCTTC CTCGCTCACT GACTCGCTGC
 3241 GCTCGGTCGT TCGGCTGCGG CGAGCGGTAT CAGCTCACTC AAAGGCGGTA ATACGGTTAT
 3301 CCACAGAATC AGGGGATAAC GCAGGAAAGA ACATGTGAGC AAAAGGCCAG CAAAAGGCCA
 3361 GGAACCGTAA AAAGGCCGCG TTGCTGGCGT TTTTCCATAG GCTCCGCCCC CCTGACGAGC
 3421 ATCACAAAAA TCGACGCTCA AGTCAGAGGT GGCGAAACCC GACAGGACTA TAAAGATACC
 3481 AGGCGTTTCC CCCTGGAAGC TCCCTCGTGC GCTCTCCTGT TCCGACCCTG CCGCTTACCG
 3541 GATACCTGTC CGCCTTTCTC CCTTCGGGAA GCGTGGCGCT TTCTCAATGC TCACGCTGTA
 3601 GGTATCTCAG TTCGGTGTAG GTCGTTCGCT CCAAGCTGGG CTGTGTGCAC GAACCCCCCG
 3661 TTCAGCCCGA CCGCTGCGCC TTATCCGGTA ACTATCGTCT TGAGTCCAAC CCGGTAAGAC
 3721 ACGACTTATC GCCACTGGCA GCAGCCACTG GTAACAGGAT TAGCAGAGCG AGGTATGTAG
 3781 GCGGTGCTAC AGAGTTCTTG AAGTGGTGGC CTAACTACGG CTACACTAGA AGGACAGTAT
 3841 TTGGTATCTG CGCTCTGCTG AAGCCAGTTA CCTTCGGAAA AAGAGTTGGT AGCTCTTGAT
 3901 CCGGCAAACA AACCACCGCT GGTAGCGGTG GTTTTTTTGT TTGCAAGCAG CAGATTACGC
 3961 GCAGAAAAAA AGGATCTCAA GAAGATCCTT TGATCTTTTC TACGGGGTCT GACGCTCAGT
 4021 GGAACGAAAA CTCACGTTAA GGGATTTTGG TCATGAGATT ATCAAAAAGG ATCTTCACCT
 4081 AGATCCTTTT AAATTAAAAA TGAAGTTTTA AATCAATCTA AAGTATATAT GAGTAAACTT
 4141 GGTCTGACAG TTACCAATGC TTAATCAGTG AGGCACCTAT CTCAGCGATC TGTCTATTTC
 4201 GTTCATCCAT AGTTGCCTGA CTCCCCGTCG TGTAGATAAC TACGATACGG GAGGGCTTAC
 4261 CATCTGGCCC CAGTGCTGCA ATGATACCGC GAGACCCACG CTCACCGGCT CCAGATTTAT
 4321 CAGCAATAAA CCAGCCAGCC GGAAGGGCCG AGCGCAGAAG TGGTCCTGCA ACTTTATCCG
 4381 CCTCCATCCA GTCTATTAAT TGTTGCCGGG AAGCTAGAGT AAGTAGTTCG CCAGTTAATA
 4441 GTTTGCGCAA CGTTGTTGCC ATTGCTACAG GCATCGTGGT GTCACGCTCG TCGTTTGGTA
 4501 TGGCTTCATT CAGCTCCGGT TCCCAACGAT CAAGGCGAGT TACATGATCC CCCATGTTGT
 4561 GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC CGATCGTTGT CAGAAGTAAG TTGGCCGCAG
 4621 TGTTATCACT CATGGTTATG GCAGCACTGC ATAATTCTCT TACTGTCATG CCATCCGTAA
 4681 GATGCTTTTC TGTGACTGGT GAGTACTCAA CCAAGTCATT CTGAGAATAG TGTATGCGGC
 4741 GACCGAGTTG CTCTTGCCCG GCGTCAATAC GGGATAATAC CGCGCCACAT AGCAGAACTT
 4801 TAAAAGTGCT CATCATTGGA AAACGTTCTT CGGGGCGAAA ACTCTCAAGG ATCTTACCGC
 4861 TGTTGAGATC CAGTTCGATG TAACCCACTC GTGCACCCAA CTGATCTTCA GCATCTTTTA
 4921 CTTTCACCAG CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA AAATGCCGCA AAAAAGGGAA
 4981 TAAGGGCGAC ACGGAAATGT TGAATACTCA TACTCTTCCT TTTTCAATAT TATTGAAGCA
 5041 TTTATCAGGG TTATTGTCTC ATGAGCGGAT ACATATTTGA ATGTATTTAG AAAAATAAAC
 5101 AAATAGGGGT TCCGCGCACA TTTCCCCGAA AAGTGCCACC TGACGTC