pAD-GAL4-2.1

编号	载体名称
YB2300	pAD-GAL4-2.1

 

pAD-GAL4-2.1载体基本信息

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。
组成型/诱导型:	组成型

pAD-GAL4-2.1载体质粒图谱和多克隆位点信息

pAD-GAL4-2.1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).
 
pAD-GAL4-2.1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