| 出品公司: | Stratagene（Agilent） |
|---|---|
| 载体名称: | pAD-GAL4-2.1 |
| 质粒类型: | 酵母双杂交载体；酵母噬粒载体 |
| 克隆方法: | 多克隆位点，限制性内切酶 |
| 启动子: | ADH1 |
| 载体大小: | 7653 bp |
| 5' 测序引物及序列: | -- |
| 3' 测序引物及序列: | -- |
| 载体标签: | 无 |
| 载体抗性: | 氨苄青霉素Ampicillin |
| 真核筛选标记: | LEU2 |
| 克隆菌株: | XL1-Blue MRF' |
| 表达菌株: | YRG-2 |
| 备注: |
含a beta-gal报告基因。 配套载体为pBD-GAL4-Cam。 |
| 产品目录号: | #838401-13 |
| 组成型/诱导型: | 组成型 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
酵母双杂交系统
Protein–protein interactions occur in many biological processes including replication, transcription, secretion, signal transduction, and metabolism. A fundamental question in the study of any protein is to identify proteins that interact with a given protein in vivo. Intense research efforts are focused on the identification of these proteins.
The GAL4 two-hybrid phagemid vector system, a eukaryotic
system to detect protein–protein interactions in vivo, provides a method for the rapid identification of genes encoding proteins that interact with a given protein (i.e., a bait protein).1,2 The system is based on the ability to separate eukaryotic transcriptional activators into two separate domains, the DNAbinding domain (BD) and the transcriptional activation domain (AD).
In theGAL4 two-hybrid phagemid vector system, proteins that interact with the bait protein are identified by generating hybrids of the yeast GAL4 BD and the bait protein (X) and the GAL4 AD and a library of proteins (Y). Neither hybrid protein is capable of initiating specific transcription of reporter genes in yeast in the absence of a specific interaction with the other hybrid protein.
When the hybrid protein X is expressed in yeast, the GAL4 BD binds X to specific DNA sequences in the yeast chromosome defined by the GAL1 or GAL4 upstream activating sequences (UASGAL1 or UASGAL4, respectively), which regulate the expression of a reporter gene. Binding of X to the UAS is not sufficient to initiate transcription of the reporter gene. When Y is expressed in yeast, the AD interacts with other components of the transcription machinery required to initiate transcription of the reporter gene. However, Y alone is not localized to the reporter gene UAS and therefore does not activate transcription of the reporter gene. When a specific interaction between X and Y localizes both the GAL4 BD and GAL4 AD to the reporter gene UAS, transcriptional activation of the reporter gene occurs (Figure 2B). The reporter genes in the GAL4 twohybrid phagemid vector system are β-galactosidase (lacZ) and histidine (HIS3).
双杂交载体pAD-GAL4-2.1和pBD-GAL4-Cam
The pAD-GAL4-2.1 phagemid vector contains a multiple cloning site (MCS) with BamH I, Nhe I, EcoR I, Xho I, Sal I, Xba I, Pst I, and Bgl II restriction sites. The pBD-GAL4-Cam phagemid vector contains an MCS with EcoR I, Srf I, Sma I, Xho I, Sal I, Xba I, and Pst I restriction sites. The unique EcoR I and Xho I cloning sites in the pAD-GAL4-2.1 vector make this vector compatible with the Agilent cDNA Synthesis Kit for the preparation of unidirectional cDNA libraries.
The unique EcoR I and Sal I cloning sites are used for the preparation of cDNA libraries in the pBD-GAL4 Cam phagemid vector because the Xho I site in the MCS is not unique. The unique BamH I, Nhe I, and EcoR I sites at the 5´ end and the Xho I, Sal I, Xba I, and Bgl II sites at the 3´ end of the DNA insert facilitate the transfer of DNA encoding the target protein into commonly used protein expression/purification vectors. Genomic DNA digested with Mbo I, BamH I, or Sau3A I and partially filled-in can be inserted into a partially filled-in Xho I site in the pAD-GAL4-2.1 phagemid vector. The Xba I site in the pAD-GAL4-2.1 and pBD-GAL4-Cam phagemid vectors is not unique and contains the UAG amber suppressor in the same translational reading frame as the GAL4 domain. DNA should therefore be inserted such that the Xba I site is not between the GAL4 domain and the DNA insert.
The pAD-GAL4-2.1 and pBD-GAL4 Cam phagemid vectors contain the pUC origin for replication and an f1 origin for production of single-stranded DNA (ssDNA) in E. coli. Single-stranded DNA can be used for DNA sequencing or site-directed mutagenesis. The pAD-GAL4-2.1 and pBDGAL4-Cam phagemid vectors contain ampicillin-resistance gene [β-lactamase (bla)] and chloramphenicol acetyltransferase genes, respectively, for selection with ampicillin and chloramphenicol in E. coli. The pADGAL4-2.1 and pBD-GAL4 Cam phagemid vectors contain the 2μ origin for replication. For selection in yeast, the pAD-GAL4-2.1 phagemid vector contains the LEU2 gene and the pBD-GAL4 Cam phagemid vector contains the TRP1 gene. The hybrid protein is expressed by the alcohol dehydrogenase 1 (ADH1) promoter (P ADH1) and is terminated by the ADH1 terminator (T ADH1).
载体序列
pAD-GAL4-2.1, 7653 bp version 122001 NOTE: The following sequence has been verified for accuracy at the junctions. The remainder of the sequence has been obtained from existing data. 1 GCTTGCATGC AACTTCTTTT CTTTTTTTTT CTTTTCTCTC TCCCCCGTTG 51 TTGTCTCACC ATATCCGCAA TGACAAAAAA AATGATGGAA GACACTAAAG 101 GAAAAAATTA ACGACAAAGA CAGCACCAAC AGATGTCGTT GTTCCAGAGC 151 TGATGAGGGG TATCTTCGAA CACACGAAAC TTTTTCCTTC CTTCATTCAC 201 GCACACTACT CTCTAATGAG CAACGGTATA CGGCCTTCCT TCCAGTTACT 251 TGAATTTGAA ATAAAAAAAG TTTGCCGCTT TGCTATCAAG TATAAATAGA 301 CCTGCAATTA TTAATCTTTT GTTTCCTCGT CATTGTTCTC GTTCCCTTTC 351 TTCCTTGTTT CTTTTTCTGC ACAATATTTC AAGCTATACC AAGCATACAA 401 TCAACTCCAA GCTTTGCAAA GATGGATAAA GCGGAATTAA TTCCCGAGCC 451 TCCAAAAAAG AAGAGAAAGG TCGAATTGGG TACCGCCGCC AATTTTAATC 501 AAAGTGGGAA TATTGCTGAT AGCTCATTGT CCTTCACTTT CACTAACAGT 551 AGCAACGGTC CGAACCTCAT AACAACTCAA ACAAATTCTC AAGCGCTTTC 601 ACAACCAATT GCCTCCTCTA ACGTTCATGA TAACTTCATG AATAATGAAA 651 TCACGGCTAG TAAAATTGAT GATGGTAATA ATTCAAAACC ACTGTCACCT 701 GGTTGGACGG ACCAAACTGC GTATAACGCG TTTGGAATCA CTACAGGGAT 751 GTTTAATACC ACTACAATGG ATGATGTATA TAACTATCTA TTCGATGATG 801 AAGATACCCC ACCAAACCCA AAAAAAGAGA TCGAATTAGG ATCCTCTGCT 851 AGCAGAGAAT TCAATTCTCT AATGCTTCTC GAGAGTATTA GTCGACTCTA 901 GAGCCCTATA GTGAGTCGTA TTACTGCAGA GATCTATGAA TCGTAGATAC 951 TGAAAAACCC CGCAAGTTCA CTTCAACTGT GCATCGTGCA CCATCTCAAT 1001 TTCTTTCATT TATACATCGT TTTGCCTTCT TTTATGTAAC TATACTCCTC 1051 TAAGTTTCAA TCTTGGCCAT GTAACCTCTG ATCTATAGAA TTTTTTAAAT 1101 GACTAGAATT AATGCCCATC TTTTTTTTGG ACCTAAATTC TTCATGAAAA 1151 TATATTACGA GGGCTTATTC AGAAGCTTTG GACTTCTTCG CCAGAGGTTT 1201 GGTCAAGTCT CCAATCAAGG TTGTCGGCTT GTCTACCTTG CCAGAAATTT 1251 ACGAAAAGAT GGAAAAGGGT CAAATCGTTG GTAGATACGT TGTTGACACT 1301 TCTAAATAAG CGAATTTCTT ATGATTTATG ATTTTTATTA TTAAATAAGT 1351 TATAAAAAAA ATAAGTGTAT ACAAATTTTA AAGTGACTCT TAGGTTTTAA 1401 AACGAAAATT CTTGTTCTTG AGTAACTCTT TCCTGTAGGT CAGGTTGCTT 1451 TCTCAGGTAT AGCATGAGGT CGCTCTTATT GACCACACCT CTACCGGCAT 1501 GCCCGAAATT CCCCTACCCT ATGAACATAT TCCATTTTGT AATTTCGTGT 1551 CGTTTCTATT ATGAATTTCA TTTATAAAGT TTATGTACAA ATATCATAAA 1601 AAAAGAGAAT CTTTTTAAGC AAGGATTTTC TTAACTTCTT CGGCGACAGC 1651 ATCACCGACT TCGGTGGTAC TGTTGGAACC ACCTAAATCA CCAGTTCTGA 1701 TACCTGCATC CAAAACCTTT TTAACTGCAT CTTCAATGGC CTTACCTTCT 1751 TCAGGCAAGT TCAATGACAA TTTCAACATC ATTGCAGCAG ACAAGATAGT 1801 GGCGATAGGG TCAACCTTAT TCTTTGGCAA ATCTGGAGCA GAACCGTGGC 1851 ATGGTTCGTA CAAACCAAAT GCGGTGTTCT TGTCTGGCAA AGAGGCCAAG 1901 GACGCAGATG GCAACAAACC CAAGGAACCT GGGATAACGG AGGCTTCATC 1951 GGAGATGATA TCACCAAACA TGTTGCTGGT GATTATAATA CCATTTAGGT 2001 GGGTTGGGTT CTTAACTAGG ATCATGGCGG CAGAATCAAT CAATTGATGT 2051 TGAACCTTCA ATGTAGGAAA TTCGTTCTTG ATGGTTTCCT CCACAGTTTT 2101 TCTCCATAAT CTTGAAGAGG CCAAAACATT AGCTTTATCC AAGGACCAAA 2151 TAGGCAATGG TGGCTCATGT TGTAGGGCCA TGAAAGCGGC CATTCTTGTG 2201 ATTCTTTGCA CTTCTGGAAC GGTGTATTGT TCACTATCCC AAGCGACACC 2251 ATCACCATCG TCTTCCTTTC TCTTACCAAA GTAAATACCT CCCACTAATT 2301 CTCTGACAAC AACGAAGTCA GTACCTTTAG CAAATTGTGG CTTGATTGGA 2351 GATAAGTCTA AAAGAGAGTC GGATGCAAAG TTACATGGTC TTAAGTTGGC 2401 GTACAATTGA AGTTCTTTAC GGATTTTTAG TAAACCTTGT TCAGGTCTAA 2451 CACTACCTGT ACCCCATTTA GGACCACCCA CAGCACCTAA CAAAACGGCA 2501 TCAACCTTCT TGGAGGCTTC CAGCGCCTCA TCTGGAAGTG GGACACCTGT 2551 AGCATCGATA GCAGCACCAC CAATTAAATG ATTTTCGAAA TCGAACTTGA 2601 CATTGGAACG AACATCAGAA ATAGCTTTAA GAACCTTAAT GGCTTCGGCT 2651 GTGATTTCTT GACCAACGTG GTCACCTGGC AAAACGACGA TCTTCTTAGG 2701 GGCAGACATT AGAATGGTAT ATCCTTGAAA TATATATATA TATTGCTGAA 2751 ATGTAAAAGG TAAGAAAAGT TAGAAAGTAA GACGATTGCT AACCACCTAT 2801 TGGAAAAAAC AATAGGTCCT TAAATAATAT TGTCAACTTC AAGTATTGTG 2851 ATGCAAGCAT TTAGTCATGA ACGCTTCTCT ATTCTATATG AAAAGCCGGT 2901 TCCGGCCTCT CACCTTTCCT TTTTCTCCCA ATTTTTCAGT TGAAAAAGGT 2951 ATATGCGTCA GGCGACCTCT GAAATTAACA AAAAATTTCC AGTCATCGAA 3001 TTTGATTCTG TGCGATAGCG CCCCTGTGTG TTCTCGTTAT GTTGAGGAAA 3051 AAAATAATGG TTGCTAAGAG ATTCGAACTC TTGCATCTTA CGATACCTGA 3101 GTATTCCCAC AGTTGGGGAT CTCGACTCTA GCTAGAGGAT CAATTCGTAA 3151 TCATGGTCAT AGCTGTTTCC TGTGTGAAAT TGTTATCCGC TCACAATTCC 3201 ACACAACATA CGAGCCGGAA GCATAAAGTG TAAAGCCTGG GGTGCCTAAT 3251 GAGTGAGGTA ACTCACATTA ATTGCGTTGC GCTCACTGCC CGCTTTCCAG 3301 TCGGGAAACC TGTCGTGCCA GTTATATGCG GCCGCATAAT TTAGGCGCGT 3351 AAATTGTAAG CGTTAATATT TTGTTAAAAT TCGCGTTAAA TTTTTGTTAA 3401 ATCAGCTCAT TTTTTAACCA ATAGGCCGAA ATCGGCAAAA TCCCTTATAA 3451 ATCAAAAGAA TAGACCGAGA TAGGGTTGAG TGTTGTTCCA GTTTGGAACA 3501 AGAGTCCACT ATTAAAGAAC GTGGACTCCA ACGTCAAAGG GCGAAAAACC 3551 GTCTATCAGG GCGATGGCCC ACTACGTGAA CCATCACCCT AATCAAGTTT 3601 TTTGGGGTCG AGGTGCCGTA AAGCACTAAA TCGGAACCCT AAAGGGAGCC 3651 CCCGATTTAG AGCTTGACGG GGAAAGCCGG CGAACGTGGC GAGAAAGGAA 3701 GGGAAGAAAG CGAAAGGAGC GGGCGCTAGG GCGCTGGCAA GTGTAGCGGT 3751 CACGCTGCGC GTAACCACCA CACCCGCCGC GCTTAATGCG CCGCTACAGG 3801 GCGCGTCAGG TGGCACTTTT CGGGGAAATG TGCGCGGAAC CCCTATTTGT 3851 TTATTTTTCT AAATACATTC AAATATGTAT CCGCTCATGA GACAATAACC 3901 CTGATAAATG CTTCAATAAT ATTGAAAAAG GAAGAGTCCT GAGGCGGAAA 3951 GAACCAGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG TCCCCAGGCT 4001 CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC 4051 AGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT 4101 GCATCTCAAT TAGTCAGCAA CCATAGTCCC GCCCCTAACT CCGCCCATCC 4151 CGCCCCTAAC TCCGCCCAGT TCCGCCCATT CTCCGCCCCA TGGCTGACTA 4201 ATTTTTTTTA TTTATGCAGA GGCCGAGGCC GCCTCGGCCA TTACAGCTGG 4251 ATTAATGAAT CGGCCAACGC GCGGGGAGAG GCGGTTTGCG TATTGGGCGC 4301 TCTTCCGCTT CCTCGCTCAC TGACTCGCTG CGCTCGGTCG TTCGGCTGCG 4351 GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA TCCACAGAAT 4401 CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC 4451 AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC 4501 CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC 4551 CGACAGGACT ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG 4601 CGCTCTCCTG TTCCGACCCT GCCGCTTACC GGATACCTGT CCGCCTTTCT 4651 CCCTTCGGGA AGCGTGGCGC TTTCTCATAG CTCACGCTGT AGGTATCTCA 4701 GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC 4751 GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA 4801 CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA 4851 TTAGCAGAGC GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG 4901 CCTAACTACG GCTACACTAG AAGGACAGTA TTTGGTATCT GCGCTCTGCT 4951 GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC 5001 AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA GCAGATTACG 5051 CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC 5101 TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT 5151 TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT 5201 AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG 5251 CTTAATCAGT GAGGCACCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA 5301 TAGTTGCCTG ACTCCCCGTC GTGTAGATAA CTACGATACG GGAGGGCTTA 5351 CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC GCTCACCGGC 5401 TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA 5451 GTGGTCCTGC AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG 5501 GAAGCTAGAG TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC 5551 CATTGCTACA GGCATCGTGG TGTCACGCTC GTCGTTTGGT ATGGCTTCAT 5601 TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TTACATGATC CCCCATGTTG 5651 TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG TCAGAAGTAA 5701 GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC 5751 TTACTGTCAT GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCA 5801 ACCAAGTCAT TCTGAGAATA GTGTATGCGG CGACCGAGTT GCTCTTGCCC 5851 GGCGTCAATA CGGGATAATA CCGCGCCACA TAGCAGAACT TTAAAAGTGC 5901 TCATCATTGG AAAACGTTCT TCGGGGCGAA AACTCTCAAG GATCTTACCG 5951 CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA ACTGATCTTC 6001 AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC 6051 AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC 6101 ATACTCTTCC TTTTTCAATA TTATTGAAGC ATTTATCAGG GTTATTGTCT 6151 CATGAGCGGA TACATATTTG AATGTATTTA GAAAAATAAA CAAATAGGGG 6201 TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC CTGACGTCTA AGAAACCATT 6251 ATTATCATGA CATTAACCTA TAAAAATAGG CGTATCACGA GGCCCTTTCG 6301 TCTCGCGCGT TTCGGTGATG ACGGTGAAAA CCTCTGACAC ATGCAGCTCC 6351 CGGAGACGGT CACAGCTTGT CTGTAAGCGG ATGCCGGGAG CAGACAAGCC 6401 CGTCAGGGCG CGTCAGCGGG TGTTGGCGGG TGTCGGGGCT GGCTTAACTA 6451 TGCGGCATCA GAGCAGATTG TACTGAGAGT GCACCATAAC GCATTTAAGC 6501 ATAAACACGC ACTATGCCGT TCTTCTCATG TATATATATA TACAGGCAAC 6551 ACGCAGATAT AGGTGCGACG TGAACAGTGA GCTGTATGTG CGCAGCTCGC 6601 GTTGCATTTT CGGAAGCGCT CGTTTTCGGA AACGCTTTGA AGTTCCTATT 6651 CCGAAGTTCC TATTCTCTAG CTAGAAAGTA TAGGAACTTC AGAGCGCTTT 6701 TGAAAACCAA AAGCGCTCTG AAGACGCACT TTCAAAAAAC CAAAAACGCA 6751 CCGGACTGTA ACGAGCTACT AAAATATTGC GAATACCGCT TCCACAAACA 6801 TTGCTCAAAA GTATCTCTTT GCTATATATC TCTGTGCTAT ATCCCTATAT 6851 AACCTACCCA TCCACCTTTC GCTCCTTGAA CTTGCATCTA AACTCGACCT 6901 CTACATTTTT TATGTTTATC TCTAGTATTA CTCTTTAGAC AAAAAAATTG 6951 TAGTAAGAAC TATTCATAGA GTGAATCGAA AACAATACGA AAATGTAAAC 7001 ATTTCCTATA CGTAGTATAT AGAGACAAAA TAGAAGAAAC CGTTCATAAT 7051 TTTCTGACCA ATGAAGAATC ATCAACGCTA TCACTTTCTG TTCACAAAGT 7101 ATGCGCAATC CACATCGGTA TAGAATATAA TCGGGGATGC CTTTATCTTG 7151 AAAAAATGCA CCCGCAGCTT CGCTAGTAAT CAGTAAACGC GGGAAGTGGA 7201 GTCAGGCTTT TTTTATGGAA GAGAAAATAG ACACCAAAGT AGCCTTCTTC 7251 TAACCTTAAC GGACCTACAG TGCAAAAAGT TATCAAGAGA CTGCATTATA 7301 GAGCGCACAA AGGAGAAAAA AAGTAATCTA AGATGCTTTG TTAGAAAAAT 7351 AGCGCTCTCG GGATGCATTT TTGTAGAACA AAAAAGAAGT ATAGATTCTT 7401 TGTTGGTAAA ATAGCGCTCT CGCGTTGCAT TTCTGTTCTG TAAAAATGCA 7451 GCTCAGATTC TTTGTTTGAA AAATTAGCGC TCTCGCGTTG CATTTTTGTT 7501 TTACAAAAAT GAAGCACAGA TTCTTCGTTG GTAAAATAGC GCTTTCGCGT 7551 TGCATTTCTG TTCTGTAAAA ATGCAGCTCA GATTCTTTGT TTGAAAAATT 7601 AGCGCTCTCG CGTTGCATTT TTGTTCTACA AAATGAAGCA CAGATGCTTC 7651 GTT
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载体质粒应用举例
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