pHIS2

pHIS2 is a reporter vector that can be used in yeast one-hybrid assays to identify and characterize DNA-binding proteins. The vector was specifically designed for use with the BD Matchmaker? One- Hybrid Library Construction & Screening Kit (#K1617-1). It contains a HIS3 nutritional reporter gene, located downstream of a multiple cloning site (MCS) and the minimal promoter of the HIS3 locus (PminHIS3). Cis-acting DNA sequences, or DNA target elements, can be inserted into the MCS and used as baits to screen GAL4 AD/cDNA fusion libraries for proteins that interact with the target sequence. A protein-DNA (or one-hybrid) interaction can be detected by performing the assay in a yeast strain such as Y187 that is auxotrophic for histidine. Positive one-hybrid interactions drive expression of the HIS3 reporter gene, which enables the host cell to grow on histidine-deficient media.

In the absence of activation, the constitutive HIS3 expression from PminHIS3 is very low. During library screening, the leaky expression of HIS3 is controlled by adding 3-amino-1,2,4-triazole (3-AT) to the medium. The concentration of 3-AT needed to fully suppress leaky HIS3 expression must be determined empirically for each DNA target element.

pHIS2 can be maintained in both yeast and bacteria. It contains an autonomous replication sequence (ARS4) and TRP1 nutritional marker for replication and selection in yeast (1, 2); it contains a Col E1 origin and a kanamycin resistance gene (Kanr) for propagation and selection in E. coli. The centromeric sequence CEN6 ensures proper segregation of the plasmid during cell division in yeast (1, 2).

载体应用

To use pHIS2 in a one-hybrid assay, clone one or more copies of a cis-acting DNA target sequence into the MCS. Then introduce the plasmid into competent yeast cells using the transformation protocol in the BD Matchmaker Library Construction & Screening Kits User Manual (PT3529-1). In contrast to the original BD Matchmaker One- Hybrid System, this reporter vector does not need to be integrated into the yeast genome. Instead, it is maintained as an episome throughout the assay. Inserting your target element may alter the level of background HIS3 expression. Therefore, constructs should be tested for background (leaky) HIS3 expression before you start a one-hybrid analysis. Background growth due to leaky HIS3 expression is controlled by adding 3-AT to the selection medium, as described in the User Manual (PT3529-1).

0001 GAATTCCCGG GGAGCTCACG CGTTCGCGAA TCGATCCGCG GTCTAGAAAT TCCTGGCATT 
0061 ATCACATAAT GAATTATACA TTATATAAAG TAATGTGATT TCTTCGAAGA ATATACTAAA 
0121 AAATGAGCAG GCAAGATAAA CGAAGGCAAA GATGACAGAG CAGAAAGCCC TAGTAAAGCG 
0181 TATTACAAAT GAAACCAAGA TTCAGATTGC GATCTCTTTA AAGGGTGGTC CCCTAGCGAT 
0241 AGAGCACTCG ATCTTCCCAG AAAAAGAGGC AGAAGCAGTA GCAGAACAGG CCACACAATC 
0301 GCAAGTGATT AACGTCCACA CAGGTATAGG GTTTCTGGAC CATATGATAC ATGCTCTGGC 
0361 CAAGCATTCC GGCTGGTCGC TAATCGTTGA GTGCATTGGT GACTTACACA TAGACGACCA 
0421 TCACACCACT GAAGACTGCG GGATTGCTCT CGGTCAAGCT TTTAAAGAGG CCCTAGGGGC 
0481 CGTGCGTGGA GTAAAAAGGT TTGGATCAGG ATTTGCGCCT TTGGATGAGG CACTTTCCAG 
0541 AGCGGTGGTA GATCTTTCGA ACAGGCCGTA CGCAGTTGTC GAACTTGGTT TGCAAAGGGA 
0601 GAAAGTAGGA GATCTCTCTT GCGAGATGAT CCCGCATTTT CTTGAAAGCT TTGCAGAGGC 
0661 TAGCAGAATT ACCCTCCACG TTGATTGTCT GCGAGGCAAG AATGATCATC ACCGTAGTGA 
0721 GAGTGCGTTC AAGGCTCTTG CGGTTGCCAT AAGAGAAGCC ACCTCGCCCA ATGGTACCAA 
0781 CGATGTTCCC TCCACCAAAG GTGTTCTTAT GTAGTGACAC CGATTATTTA AAGCTGCAGC 
0841 ATACGATATA TATACATGTG TATATATGTA TACCTATGAA TGTCAGTAAG TATGTATACG 
0901 AACAGTATGA TACTGAAGAT GACAAGGTAA TGCATCATTC TATACGTGTC ATTCTGAACG 
0961 AGGCGCGCTT TCCTTTTTTC TTTTTGCTTT TTCTTTTTTT TTCTCTTGAA CTCGAGAAAA 
1021 AAAATATAAA AGAGATGGAG GAACGGGAAA AAGTTAGTTG TGGTGATAGG TGGCAAGTGG 
1081 TATTCCGTAA GAACAACAAG AAAAGCATTT CATATTATGG CTGAACTGAG CGAACAAGTG 
1141 CAAAATTTAA GCATCAACGA CAACAACGAG AATGGTTATG TTCCTCCTCA CTTAAGAGGA 
1201 AAACCAAGAA GTGCCAGAAA TAACATGAGC AACTACAATA ACAACAACGG CGGCTACAAC 
1261 GGTGGCCGTG GCGGTGGCAG CTTCTTTAGC AACAACCGTC GTGGTGGTTA CGGCAACGGT 
1321 GGTTTCTTCG GTGGAAACAA CGGTGGCAGC AGATCTAACG GCCGTTCTGG TGGTAGATGG 
1381 ATCGATGGCA AACATGTCCC AGCTCCAAGA AACGAAAAGG CCGAGATCGC CATATTTGGT 
1441 GTCCCCGAGG ATCCTCTACG CCGGACGCAT CGTGGCCGGC ATCACCGGCG CCACAGGTGC 
1501 GGTTGCTGGC GCCTATATCG CCGACATCAC CGATGGGGAA GATCGGGCTC GCCACTTCGG 
1561 GCTCATGAGC GCTTGTTTCG GCGTGGGTAT GGTGGCAGGC CCCGTGGCCG GGGGACTGTT 
1621 GGGCGCCATC TCCTTGCATG CACCATTCCT TGCGGCGGCG GTGCTCAACG GCCTCAACCT 
1681 ACTACTGGGC TGCTTCCTAA TGCAGGAGTC GCATAAGGGA GAGCGTCGAC CGATGCCCTT 
1741 GAGAGCCTTC AACCCAGTCA GCTCCTTCCG GTGGGCGCGG GGCATGACTA TCGTCGCCGC 
1801 ACTTATGACT GTCTTCTTTA TCATGCAACT CGTAGGACAG GTGCCGGCAG CGCTCTGGGT 
1861 CATTTTCGGC GAGGACCGCT TTCGCTGGAG CGCGACGATG ATCGGCCTGT CGCTTGCGGT 
1921 ATTCGGAATC TTGCACGCCC TCGCTCAAGC CTTCGTCACT GGTCCCGCCA CCAAACGTTT 
1981 CGGCGAGAAG CAGGCCATTA TCGCCGGCAT GGCGGCCGAC GCGCTGGGCT ACGTCTTGCT 
2041 GGCGTTCGCG ACGCGAGGCT GGATGGCCTT CCCCATTATG ATTCTTCTCG CTTCCGGCGG 
2101 CATCGGGATG CCCGCGTTGC AGGCCATGCT GTCCAGGCAG GTAGATGACG ACCATCAGGG 
2161 ACAGCTTCAA GGATCGCTCG CGGCTCTTAC CAGCCTAACT TCGATCATTG GACCGCTGAT 
2221 CGTCACGGCG ATTTATGCCG CCTCGGCGAG CACATGGAAC GGGTTGGCAT GGATTGTAGG 
2281 CGCCGCCCTA TACCTTGTCT GCCTCCCCGC GTTGCGTCGC GGTGCATGGA GCCGGGCCAC 
2341 CTCGACCTGA ATGGAAGCCG GCGGCACCTC GCTAACGGAT TCACCACTCC AAGAATTGGA 
2401 GCCAATCAAT TCTTGCGGAG AACTGTGAAT GCGCAAACCA ACCCTTGGCA GAACATATCC 
2461 ATCGCGTCCG CCATCTCCAG CAGCCGCACG CGGCGCATCG GGGGGGGGGT TTCAATTCAA 
2521 TTCATCATTT TTTTTTTATT CTTTTTTTTG ATTTCGGTTT CTTTGAAATT TTTTTAACGA 
2581 CATTACTATA TATATAATAT AGGAAGCATT TAATAGAACA GCATCGTAAT ATATGTGTAC 
2641 TTTGCAGTTA TGACGCCAGA TGGCAGTAGT GGAAGATATT CTTTATTGAA AAATAGCTTG 
2701 TCACCTTACG TACAATCTTG ATCCGGAGCT TTTCTTTTTT TGCCGATTAA GAATTAATTC 
2761 GGTCGAAAAA AGAAAAGGAG AGGGCCAAGA GGGAGGGCAT TGGTGACTAT TGAGCACGTG 
2821 AGTATACGTG ATTAAGCACA CAAAGGCAGC TTGGAGTATG TCTGTTATTA ATTTCACAGG 
2881 TAGTTCTGGT CCATTGGTGA AAGTTTGCGG CTTGCAGAGC ACAGAGGCCG CAGAATGTGC 
2941 TCTAGATTCC GATGCTGACT TGCTGGGTAT TATATGTGTG CCCAATAGAA AGAGAACAAT 
3001 TGACCCGGTT ATTGCAAGGA AAATTTCAAG TCTTGTAAAA GCATATAAAA ATAGTTCAGG 
3061 CACTCCGAAA TACTTGGTTG GCGTGTTTCG TAATCAACCT AAGGAGGATG TTTTGGCTCT 
3121 GGTCAATGAT TACGGCATTG ATATCGTCCA ACTGCATGGA GATGAGTCGT GGCAAGAATA 
3181 CCAAGAGTTC CTCGGTTTGC CAGTTATTAA AAGACTCGTA TTTCCAAAAG ACTGCAACAT 
3241 ACTACTCAGT GCAGCTTCAC AGAAACCTCA TTCGTTTATT CCCTTGTTTG ATTCAGAAGC 
3301 AGGTGGGACA GGTGAACTTT TGGATTGGAA CTCGATTTCT GACTGGGTTG GAAGGCAAGA 
3361 GAGCCCCGAA AGCTTACATT TTATGTTAGC TGGTGGACTG ACGCCAGAAA ATGTTGGTGA 
3421 TGCGCTTAGA TTAAATGGCG TTATTGGTGT TGATGTAAGC GGAGGTGTGG AGACAAATGG 
3481 TGTAAAAGAC TCTAACAAAA TAGCAAATTT CGTCAAAAAT GCTAAGAAAT AGGTTATTAC 
3541 TGAGTAGTAT TTATTTAAGT ATTGTTTGTG CACTTGCCCT AGAGTCGACC TGCAGGCATG 
3601 CAAGCTTTTG TTCCCTTTAG TGAGGGTTAA TTTCGAGCTT GGCGTAATCA TGGTCATAGC 
3661 TGTTTCCTGT GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA 
3721 TAAAGTGTAA AGCCTGGGGT GCCTAATGAG TGAGCTAACT CACATTAATT GCGTTGCGCT 
3781 CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA ATCGGCCAAC 
3841 GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC 
3901 TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT 
3961 TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC CAGCAAAAGG 
4021 CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG 
4081 AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT 
4141 ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA 
4201 CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT 
4261 GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC 
4321 CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA 
4381 GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG 
4441 TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGGACAG 
4501 TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT 
4561 GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA 
4621 CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC 
4681 AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA 
4741 CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA 
4801 CTTGGTCTGA CAGTCAGAAG AACTCGTCAA GAAGGCGATA GAAGGCGATG CGCTGCGAAT 
4861 CGGGAGCGGC GATACCGTAA AGCACGAGGA AGCGGTCAGC CCATTCGCCG CCAAGCTCTT 
4921 CAGCAATATC ACGGGTAGCC AACGCTATGT CCTGATAGCG GTCCGCCACA CCCAGCCGGC 
4981 CACAGTCGAT GAATCCAGAA AAGCGGCCAT TTTCCACCAT GATATTCGGC AAGCAGGCAT 
5041 CGTCATGGGT CACGACGAGA TCCTCGCCGT CGGGCATGCT CGCCTTGAGC CTGGCGAACA 
5101 GTTCGGCTGG CGCGAGCCCC TGATGCTCTT CGTCCAGATC ATCCTGATCG ACAAGACCGG 
5161 CTTCCATCCG AGTACGTGCT CGCTCGATGC GATGTTTCGC TTGGTGGTCG AATGGGCAGG 
5221 TAGCCGGATC AAGCGTATGC AGCCGCCGCA TTGCATCAGC CATGATGGAT ACTTTCTCGG 
5281 CAGGAGCAAG GTGAGATGAC AGGAGATCCT GCCCCGGCAC TTCGCCCAAT AGCAGCCAGT 
5341 CCCTTCCCGC TTCAGTGACA ACGTCGAGCA CAGCTGCGCA AGGAACGCCC GTCGTGGCCA 
5401 GCCACGATAG CCGCGCTGCC TCGTCTTGCA GTTCATTCAG GGCACCGGAC AGGTCGGTCT 
5461 TGACAAAAAG AACCGGGCGC CCCTGCGCTG ACAGCCGGAA CACGGCGGCA TCAGAGCAGC 
5521 CGATTGTCTG TTGTGCCCAG TCATAGCCGA ATAGCCTCTC CACCCAAGCG GCCGGAGAAC 
5581 CTGCGTGCAA TCCATCTTGT TCAATCATAC TCTTCCTTTT TCAATATTAT TGAAGCATTT 
5641 ATCAGGGTTA TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA AATAAACAAA 
5701 TAGGGGTTCC GCGCACATTT CCCCGAAAAG TGCCACCTGC GGACGGATCG CTTGCCTGTA 
5761 ACTTACACGC GCCTCGTATC TTTTAATGAT GGAATAATTT GGGAATTTAC TCTGTGTTTA 
5821 TTTATTTTTA TGTTTTGTAT TTGGATTTTA GAAAGTAAAT AAAGAGTAGA AGAGTTACGG 
5881 AATGAAGAAA AAAAAATAAA CAAAGGTTTA AAAAATTTCA ACAAAAAGCG TACTTTACAT 
5941 ATATATTTAT TAGACAAGAA AAGCAGATTA AATAGATATA CATTCGATTA ACGATAAGTA 
6001 AAATGTAAAA TCACAGGATT TTCGTGTGTG GTCTTCTACA CAGACAAGAT GAAACAATTC 
6061 GGCATTAATA CCTGAGAGCA GGAAGAGCAA GATAAAAGGN AGTATTTGTT GGCGATCCCC 
6121 CTAGAGTCTT TTACATCTTC GGAAAACAAA AACTATTTTT TCTTTAATTT CTTTTTTTAC 
6181 TTTCTATTTT TAATTTATAT ATTTATATTA AAAAATTTAA ATTATAATTA TTTTTATAGC 
6241 ACGTGATGAA AAGGACCGAC GTCTAAGAAA CCATTATTAT CATGACATTA ACCTATAAAA 
6301 ATAGGCGTAT CACGAGGCCC TTTCGTCTCG CGCGTTTCGG TGATGACGGT GAAAACCTCT 
6361 GACACATGCA GCTCCCGGAG ACGGTCACAG CTTGTCTGTA AGCGGATGCC GGGAGCAGAC 
6421 AAGCCCGTCA GGGCGCGTCA GCGGGTGTTG GCGGGTGTCG GGGCTGGCTT AACTATGCGG 
6481 CATCAGAGCA GATTGTACTG AGAGTGCACC ATATGCGGTG TGAAATACCG CACAGATGCG 
6541 TAAGGAGAAA ATACCGCATC AGGAAATTGT AAACGTTAAT ATTTTGTTAA AATTCGCGTT 
6601 AAATTTTTGT TAAATCAGCT CATTTTTTAA CCAATAGGCC GAAATCGGCA AAATCCCTTA 
6661 TAAATCAAAA GAATAGACCG AGATAGGGTT GAGTGTTGTT CCAGTTTGGA ACAAGAGTCC 
6721 ACTATTAAAG AACGTGGACT CCAACGTCAA AGGGCGAAAA ACCGTCTATC AGGGCGATGG 
6781 CCCACTACGT GAACCATCAC CCTAATCAAG TTTTTTGGGG TCGAGGTGCC GTAAAGCACT 
6841 AAATCGGAAC CCTAAAGGGA GCCCCCGATT TAGAGCTTGA CGGGGAAAGC CGGCGAACGT 
6901 GGCGAGAAAG GAAGGGAAGA AAGCGAAAGG AGCGGGCGCT AGGGCGCTGG CAAGTGTAGC 
6961 GGTCACGCTG CGCGTAACCA CCACACCCGC CGCGCTTAAT GCGCCGCTAC AGGGCGCGTC 
7021 GCGCCATTCG CCATTCAGGC TGCGCAACTG TTGGGAAGGG CGATCGGTGC GGGCCTCTTC 
7081 GCTATTACGC CAGCTGGCGA AAGGGGGATG TGCTGCAAGG CGATTAAGTT GGGTAACGCC 
7141 AGGGTTTTCC CAGTCACGAC GTTGTAAAAC GACGGCCAGT GAATTGTAAT ACGACTCACT 
7201 ATAGGGC