pBD-GAL4-Cam
pBD-GAL4-Cam
pBD-GAL4-Cam
编号 | 载体名称 |
北京华越洋生物VECT2870 | pBD-GAL4-Cam |
pBD-GAL4-Cam载体基本信息
| 载体名称: | pBD-GAL4-Cam |
|---|---|
| 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: | 6491 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: | 氯霉素Chloramphenicol |
| 真核筛选标记: | TRP1 |
| 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pAD-GAL4-2.1 |
| 产品目录号: | -- |
| 组成型/诱导型: | 组成型 |
pBD-GAL4-Cam载体质粒图谱和多克隆位点信息
pBD-GAL4-Cam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-CamThe 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).
pBD-GAL4-Cam载体序列
pBD-GAL4-Cam, 6491 bp version 045002 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 ATGATGGAAG ACACTAAAGG 101 AAAAAATTAA CGACAAAGAC AGCACCAACA GATGTCGTTG TTCCAGAGCT 151 GATGAGGGGT ATCTCGAAGC ACACGAAACT TTTTCCTTCC TTCATTCACG 201 CACACTACTC TCTAATGAGC AACGGTATAC GGCCTTCCTT CCAGTTACTT 251 GAATTTGAAA TAAAAAAAAG TTTGCTGTCT TGCTATCAAG TATAAATAGA 301 CCTGCAATTA TTAATCTTTT GTTTCCTCGT CATTGTTCTC GTTCCCTTTC 351 TTCCTTGTTT CTTTTTCTGC ACAATATTTC AAGCTATACC AAGCATACAA 401 TCAACTCCAA GCTTGAAGCA AGCCTCCTGA AAGATGAAGC TACTGTCTTC 451 TATCGAACAA GCATGCGATA TTTGCCGACT TAAAAAGCTC AAGTGCTCCA 501 AAGAAAAACC GAAGTGCGCC AAGTGTCTGA AGAACAACTG GGAGTGTCGC 551 TACTCTCCCA AAACCAAAAG GTCTCCGCTG ACTAGGGCAC ATCTGACAGA 601 AGTGGAATCA AGGCTAGAAA GACTGGAACA GCTATTTCTA CTGATTTTTC 651 CTCGAGAAGA CCTTGACATG ATTTTGAAAA TGGATTCTTT ACAGGATATA 701 AAAGCATTGT TAACAGGATT ATTTGTACAA GATAATGTGA ATAAAGATGC 751 CGTCACAGAT AGATTGGCTT CAGTGGAGAC TGATATGCCT CTAACATTGA 801 GACAGCATAG AATAAGTGCG ACATCATCAT CGGAAGAGAG TAGTAACAAA 851 GGTCAAAGAC AGTTGACTGT ATCGCCGGAA TTCGCCCGGG CCTCGAGCCC 901 GGGTCGACTC TAGAGCCCTA TAGTGAGTCG TATTACTGCA GCCAAGCTAA 951 TTCCGGGCGA ATTTCTTATG ATTTATGATT TTTATTATTA AATAAGTTAT 1001 AAAAAAAATA AGTGTATACA AATTTTAAAG TGACTCTTAG GTTTTAAAAC 1051 GAAAATTCTT GTTCTTGAGT AACTCTTTCC TGTAGGTCAG GTTGCTTTCT 1101 CAGGTATAGC ATGAGGTCGC TCTTATTGAC CACACCTCTA CCGGCATGCC 1151 GGCAAGTGCA CAAACAATAC TTAAATAAAT ACTACTCAGT AATAACCTAT 1201 TTCTTAGCAT TTTTGACGAA ATTTGCTATT TTGTTAGAGT CTTTTACACC 1251 ATTTGTCTCC ACACCTCCGC TTACATCAAC ACCAATAACG CCATTTAATC 1301 TAAGCGCATC ACCAACATTT TCTGGCGTCA GTCCACCAGC TAACATAAAA 1351 TGTAAGCTTT CGGGGCTCTC TTGCCTTCCA ACCCAGTCAG AAATCGAGTT 1401 CCAATCCAAA AGTTCACCTG TCCCACCTGC TTCTGAATCA AACAAGGGAA 1451 TAAACGAATG AGGTTTCTGT GAAGCTGCAC TGAGTAGTAT GTTGCAGTCT 1501 TTTGGAAATA CGAGTCTTTT AATAACTGGC AAACCGAGGA ACTCTTGGTA 1551 TTCTTGCCAC GACTCATCTC CATGCAGTTG GACGATATCA ATGCCGTAAT 1601 CATTGACCAG AGCCAAAACA TCCTCCTTAG GTTGATTACG AAACACGCCA 1651 ACCAAGTATT TCGGAGTGCC TGAACTATTT TTATATGCTT TTACAAGACT 1701 TGAAATTTTC CTTGCAATAA CCGGGTCAAT TGTTCTCTTT CTATTGGGCA 1751 CACATATAAT ACCCAGCAAG TCAGCATCGG AATCTAGAGC ACATTCTGCG 1801 GCCTCTGTGC TCTGCAAGCC GCAAACTTTC ACCAATGGAC CAGAACTACC 1851 TGTGAAATTA ATAACAGACA TACTCCAAGC TGCCTTTGTG TGCTTAATCA 1901 CGTATACTCA CGTGCTCAAT AGTCACCAAT GCCCTCCCTC TTGGCCCTCT 1951 CCTTTTCTTT TTTCGACCGA ATTAATTCGT AATCATGGTC ATAGCTGTTT 2001 CCTGTGTGAA ATTGTTATCC GCTCACAATT CCACACAACA TACGAGCCGG 2051 AAGCATAAAG TGTAAAGCCT GGGGTGCCTA ATGAGTGAGG TAACTCACAT 2101 TAATTGCGTT GCGCTCACTG CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC 2151 CAGCTGGTTC TTTCCGCCTC AGGACTCTTC CTTTTTCAAT ATTATTGAAG 2201 CATTTATCAG GGTTATTGTC TCATGAGCGG ATACATATTT GAATGTATTT 2251 AGAAAAATAA ACAAATAGGG GTTCCGCGCA CATTTCCCCG AAAAGTGCCA 2301 CCTGACGCGC CCTGTAGCGG CGCATTAAGC GCGGCGGGTG TGGTGGTTAC 2351 GCGCAGCGTG ACCGCTACAC TTGCCAGCGC CCTAGCGCCC GCTCCTTTCG 2401 CTTTCTTCCC TTCCTTTCTC GCCACGTTCG CCGGCTTTCC CCGTCAAGCT 2451 CTAAATCGGG GGCTCCCTTT AGGGTTCCGA TTTAGTGCTT TACGGCACCT 2501 CGACCCCAAA AAACTTGATT AGGGTGATGG TTCACGTAGT GGGCCATCGC 2551 CCTGATAGAC GGTTTTTCGC CCTTTGACGT TGGAGTCCAC GTTCTTTAAT 2601 AGTGGACTCT TGTTCCAAAC TGGAACAACA CTCAACCCTA TCTCGGTCTA 2651 TTCTTTTGAT TTATAAGGGA TTTTGCCGAT TTCGGCCTAT TGGTTAAAAA 2701 ATGAGCTGAT TTAACAAAAA TTTAACGCGA ATTTTAACAA AATATTAACG 2751 CTTACAATTT ACGCGCCTAA ATTATGCGGC CGCATATAAC TGGATTAATG 2801 AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG CGCTCTTCCG 2851 CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG 2901 GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA 2951 TAACGCAGGA AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC 3001 GTAAAAAGGC CGCGTTGCTG GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC 3051 GAGCATCACA AAAATCGACG CTCAAGTCAG AGGTGGCGAA ACCCGACAGG 3101 ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC GTGCGCTCTC 3151 CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG 3201 GGAAGCGTGG CGCTTTCTCA TAGCTCACGC TGTAGGTATC TCAGTTCGGT 3251 GTAGGTCGTT CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC 3301 CCGACCGCTG CGCCTTATCC GGTAACTATC GTCTTGAGTC CAACCCGGTA 3351 AGACACGACT TATCGCCACT GGCAGCAGCC ACTGGTAACA GGATTAGCAG 3401 AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG TGGCCTAACT 3451 ACGGCTACAC TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA 3501 GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC 3551 CGCTGGTAGC GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA 3601 AAAAAGGATC TCAAGAAGAT CCTTTGATCT TTTCTACGGG GTCTGACGCT 3651 CAGTGGAACG AAAACTCACG TTAAGGGATT TTGGTCATGA GATTATCAAA 3701 AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT TTTAAATCAA 3751 TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC 3801 AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC 3851 CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG 3901 GCCCCAGTGC TGCAATGATA CCGCGAGACC CACGCTCACC GGCTCCAGAT 3951 TTATCAGCAA TAAACCAGCC AGCCGCCTGT GACGGAAGAT CACTTCGCAG 4001 AATAAATAAA TCCTGGTGTC CCTGTTGATA CCGGGAAGCC CTGGGCCAAC 4051 TTTTGGCGAA AATGAGACGT TGATCGGCAC GTAAGAGGTT CCAACTTTCA 4101 CCATAATGAA ATAAGATCAC TACCGGGCGT ATTTTTTGAG TTGTCGAGAT 4151 TTTCAGGAGC TAAGGAAGCT AAAATGGAGA AAAAAATCAC TGGATATACC 4201 ACCGTTGATA TATCCCAATG GCATCGTAAA GAACATTTTG AGGCATTTCA 4251 GTCAGTTGCT CAATGTACCT ATAACCAGAC CGTTCAGCTG GATATTACGG 4301 CCTTTTTAAA GACCGTAAAG AAAAATAAGC ACAAGTTTTA TCCGGCCTTT 4351 ATTCACATTC TTGCCCGCCT GATGAATGCT CATCCGGAAT TACGTATGGC 4401 AATGAAAGAC GGTGAGCTGG TGATATGGGA TAGTGTTCAC CCTTGTTACA 4451 CCGTTTTCCA TGAGCAAACT GAAACGTTTT CATCGCTCTG GAGTGAATAC 4501 CACGACGATT TCCGGCAGTT TCTACACATA TATTCGCAAG ATGTGGCGTG 4551 TTACGGTGAA AACCTGGCCT ATTTCCCTAA AGGGTTTATT GAGAATATGT 4601 TTTTCGTCTC AGCCAATCCC TGGGTGAGTT TCACCAGTTT TGATTTAAAC 4651 GTGGCCAATA TGGACAACTT CTTCGCCCCG TTTTCACCAT GGGCAAATAT 4701 TATACGCAAG GCGACAAGGT GCTGATGCCG CTGGCGATTC AGGTTCATCA 4751 TGCCGTTTGT GATGGCTTCC ATGTCGGCAG AATGCTTAAT GAATTACAAC 4801 AGTACTGCGA TGAGTGGCAG GGCGGGGCGT AATTTTTTTA AGGCAGTTAT 4851 TGGTGCCCTT AAACGCCTGG TTGCTACGCC TGAATAAGTG ATAATAAGCG 4901 GATGAATGGC AGAAATTCGA AAGCAAATTC GACCCGGTCG TCGGTTCAGG 4951 GCAGGGTCGT TAAATAGCCG CTTATGTCTA TTGCTGGTTT ACCGGTTTAT 5001 TGACTACCGG AAGCAGTGTG ACCGTGTGCT TCTCAAATGC CTGAGGCCAG 5051 TTTGCTCAGG CTCTCCCCGT GGAGGCTAAG AAACCATTAT TATCATGACA 5101 TTAACCTATA AAAATAGGCG TATCACGAGG CCCTTTCGTC TCGCGCGTTT 5151 CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA 5201 CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG 5251 TCAGCGGGTG TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA 5301 GCAGATTGTA CTGAGAGTGC ACCATAACGC ATTTAAGCAT AAACACGCAC 5351 TATGCCGTTC TTCTCATGTA TATATATATA CAGGCAACAC GCAGATATAG 5401 GTGCGACGTG AACAGTGAGC TGTATGTGCG CAGCTCGCGT TGCATTTTCG 5451 GAAGCGCTCG TTTTCGGAAA CGCTTTGAAG TTCCTATTCC GAAGTTCCTA 5501 TTCTCTAGCT AGAAAGTATA GGAACTTCAG AGCGCTTTTG AAAACCAAAA 5551 GCGCTCTGAA GACGCACTTT CAAAAAACCA AAAACGCACC GGACTGTAAC 5601 GAGCTACTAA AATATTGCGA ATACCGCTTC CACAAACATT GCTCAAAAGT 5651 ATCTCTTTGC TATATATCTC TGTGCTATAT CCCTATATAA CCTACCCATC 5701 CACCTTTCGC TCCTTGAACT TGCATCTAAA CTCGACCTCT ACATTTTTTA 5751 TGTTTATCTC TAGTATTACT CTTTAGACAA AAAAATTGTA GTAAGAACTA 5801 TTCATAGAGT GAATCGAAAA CAATACGAAA ATGTAAACAT TTCCTATACG 5851 TAGTATATAG AGACAAAATA GAAGAAACCG TTCATAATTT TCTGACCAAT 5901 GAAGAATCAT CAACGCTATC ACTTTCTGTT CACAAAGTAT GCGCAATCCA 5951 CATCGGTATA GAATATAATC GGGGATGCCT TTATCTTGAA AAAATGCACC 6001 CGCAGCTTCG CTAGTAATCA GTAAACGCGG GAAGTGGAGT CAGGCTTTTT 6051 TTATGGAAGA GAAAATAGAC ACCAAAGTAG CCTTCTTCTA ACCTTAACGG 6101 ACCTACAGTG CAAAAAGTTA TCAAGAGACT GCATTATAGA GCGCACAAAG 6151 GAGAAAAAAA GTAATCTAAG ATGCTTTGTT AGAAAAATAG CGCTCTCGGG 6201 ATGCATTTTT GTAGAACAAA AAAGAAGTAT AGATTCTTTG TTGGTAAAAT 6251 AGCGCTCTCG CGTTGCATTT CTGTTCTGTA AAAATGCAGC TCAGATTCTT 6301 TGTTTGAAAA ATTAGCGCTC TCGCGTTGCA TTTTTGTTTT ACAAAAATGA 6351 AGCACAGATT CTTCGTTGGT AAAATAGCGC TTTCGCGTTG CATTTCTGTT 6401 CTGTAAAAAT GCAGCTCAGA TTCTTTGTTT GAAAAATTAG CGCTCTCGCG 6451 TTGCATTTTT GTTCTACAAA ATGAAGCACA GATGCTTCGT T
