pACT

The pACT Vector is a high-copy plasmid in which the human cytomegalovirus (CMV) immediate early promoter drives expression of the herpes virus VP16 activation domain (amino acids 411–456). A chimeric intron is located 5? of the gene segment, and a multiple cloning region is located 3? of the gene segment for insertion of cDNA clones of interest (Figures 2 and 3). The presence of this chimeric intron, in context with the CMV promoter and polyadenylation signal, can result in increased protein expression of cDNA genes linked to these elements (6). The stop codons and SV40 late polyadenylation region, demonstrated to be efficient for mRNA production (7), are at the 3? end of the fusion gene. The fusion gene region is flanked by T7 and T3 RNA polymerase promoters for the synthesis of sense and antisense RNA products. The T7 promoter allows the construct to be translated using the TNT? T7 Quick Coupled Transcription/Translation System (Cat.# L1170). Also located on this vector is the neomycin phosphotransferase gene (from Tn5) driven by the SV40 early promoter and followed by a synthetic polyadenylation cassette. This gene confers resistance to the neomycin analog, G418 (Geneticin?; 8). The plasmid backbone contains an f1 origin of replication for the production of single-stranded DNA (ssDNA) and the β-lactamase (Ampr) gene for selection of the vector DNA in E. coli.

0001 TCAATATTGG CCATTAGCCA TATTATTCAT TGGTTATATA GCATAAATCA ATATTGGCTA 
0061 TTGGCCATTG CATACGTTGT ATCTATATCA TAATATGTAC ATTTATATTG GCTCATGTCC 
0121 AATATGACCG CCATGTTGGC ATTGATTATT GACTAGTTAT TAATAGTAAT CAATTACGGG 
0181 GTCATTAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG TAAATGGCCC 
0241 GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT 
0301 AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC 
0361 CCACTTGGCA GTACATCAAG TGTATCATAT GCCAAGTCCG CCCCCTATTG ACGTCAATGA 
0421 CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC TTACGGGACT TTCCTACTTG 
0481 GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAC 
0541 CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT 
0601 CAATGGGAGT TTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTG 
0661 CGATCGCCCG CCCCGTTGAC GCAAATGGGC GGTAGGCGTG TACGGTGGGA GGTCTATATA 
0721 AGCAGAGCTC GTTTAGTGAA CCGTCAGATC ACTAGAAGCT TTATTGCGGT AGTTTATCAC 
0781 AGTTAAATTG CTAACGCAGT CAGTGCTTCT GACACAACAG TCTCGAACTT AAGCTGCAGT 
0841 GACTCTCTTA AGGTAGCCTT GCAGAAGTTG GTCGTGAGGC ACTGGGCAGG TAAGTATCAA 
0901 GGTTACAAGA CAGGTTTAAG GAGACCAATA GAAACTGGGC TTGTCGAGAC AGAGAAGACT 
0961 CTTGCGTTTC TGATAGGCAC CTATTGGTCT TACTGACATC CACTTTGCCT TTCTCTCCAC 
1021 AGGTGTCCAC TCCCAGTTCA ATTACAGCTC TTAAGGCTAG AGTACTTAAT ACGACTCACT 
1081 ATAGGCTAGC CAGCTTGAAG CAAGCCTCCT GAAAGATGAA GCTACTGTCT TCTATCGAAC 
1141 AAGCATGCCC AAAAAAGAAG AGAAAGGTAG ATGAATTCCC GGGGATCTCG ACGGCCCCCC 
1201 CGACCGATGT CAGCCTGGGG GACGAGCTCC ACTTAGACGG CGAGGACGTG GCGATGGCGC 
1261 ATGCCGACGC GCTAGACGAT TTCGATCTGG ACATGTTGGG GGACGGGGAT TCCCCGGGTC 
1321 CGGGATCGCC AGGGATCCGT CGACTTGACG CGTTGATATC ATCTAGAGCG GCCGCAGGTA 
1381 CCTGAATAAC TAAGGCCGCT TCCCTTTAGT GAGGGTTAAT GCTTCGAGCA GACATGATAA 
1441 GATACATTGA TGAGTTTGGA CAAACCACAA CTAGAATGCA GTGAAAAAAA TGCTTTATTT 
1501 GTGAAATTTG TGATGCTATT GCTTTATTTG TAACCATTAT AAGCTGCAAT AAACAAGTTA 
1561 ACAACAACAA TTGCATTCAT TTTATGTTTC AGGTTCAGGG GGAGATGTGG GAGGTTTTTT 
1621 AAAGCAAGTA AAACCTCTAC AAATGTGGTA AAATCCGATA AGGATCGATT CCGGAGCCTG 
1681 AATGGCGAAT GGACGCGCCC TGTAGCGGCG CATTAAGCGC GGCGGGTGTG GTGGTTACGC 
1741 GCAGCGTGAC CGCTACACTT GCCAGCGCCC TAGCGCCCGC TCCTTTCGCT TTCTTCCCTT 
1801 CCTTTCTCGC CACGTTCGCC GGCTTTCCCC GTCAAGCTCT AAATCGGGGG CTCCCTTTAG 
1861 GGTTCCGATT TAGTGCTTTA CGGCACCTCG ACCCCAAAAA ACTTGATTAG GGTGATGGTT 
1921 CACGTAGTGG GCCATCGCCC TGATAGACGG TTTTTCGCCC TTTGACGTTG GAGTCCACGT 
1981 TCTTTAATAG TGGACTCTTG TTCCAAACTG GAACAACACT CAACCCTATC TCGGTCTATT 
2041 CTTTTGATTT ATAAGGGATT TTGCCGATTT CGGCCTATTG GTTAAAAAAT GAGCTGATTT 
2101 AACAAAAATT TAACGCGAAT TTTAACAAAA TATTAACGCT TACAATTTCG CCTGTGTACC 
2161 TTCTGAGGCG GAAAGAACCA GCTGTGGAAT GTGTGTCAGT TAGGGTGTGG AAAGTCCCCA 
2221 GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCAGGTGT 
2281 GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA 
2341 GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC CAGTTCCGCC 
2401 CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG CAGAGGCCGA GGCCGCCTCG 
2461 GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA 
2521 AAGCTTGATT CTTCTGACAC AACAGTCTCG AACTTAAGGC TAGAGCCACC ATGATTGAAC 
2581 AAGATGGATT GCACGCAGGT TCTCCGGCCG CTTGGGTGGA GAGGCTATTC GGCTATGACT 
2641 GGGCACAACA GACAATCGGC TGCTCTGATG CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC 
2701 GCCCGGTTCT TTTTGTCAAG ACCGACCTGT CCGGTGCCCT GAATGAACTG CAGGACGAGG 
2761 CAGCGCGGCT ATCGTGGCTG GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG 
2821 TCACTGAAGC GGGAAGGGAC TGGCTGCTAT TGGGCGAAGT GCCGGGGCAG GATCTCCTGT 
2881 CATCTCACCT TGCTCCTGCC GAGAAAGTAT CCATCATGGC TGATGCAATG CGGCGGCTGC 
2941 ATACGCTTGA TCCGGCTACC TGCCCATTCG ACCACCAAGC GAAACATCGC ATCGAGCGAG 
3001 CACGTACTCG GATGGAAGCC GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG 
3061 GGCTCGCGCC AGCCGAACTG TTCGCCAGGC TCAAGGCGCG CATGCCCGAC GGCGAGGATC 
3121 TCGTCGTGAC CCATGGCGAT GCCTGCTTGC CGAATATCAT GGTGGAAAAT GGCCGCTTTT 
3181 CTGGATTCAT CGACTGTGGC CGGCTGGGTG TGGCGGACCG CTATCAGGAC ATAGCGTTGG 
3241 CTACCCGTGA TATTGCTGAA GAGCTTGGCG GCGAATGGGC TGACCGCTTC CTCGTGCTTT 
3301 ACGGTATCGC CGCTCCCGAT TCGCAGCGCA TCGCCTTCTA TCGCCTTCTT GACGAGTTCT 
3361 TCTGAGCGGG ACTCTGGGGT TCGAAATGAC CGACCAAGCG ACGCCCAACC TGCCATCACG 
3421 ATGGCCGCAA TAAAATATCT TTATTTTCAT TACATCTGTG TGTTGGTTTT TTGTGTGAAG 
3481 ATCCGCGTAT GGTGCACTCT CAGTACAATC TGCTCTGATG CCGCATAGTT AAGCCAGCCC 
3541 CGACACCCGC CAACACCCGC TGACGCGCCC TGACGGGCTT GTCTGCTCCC GGCATCCGCT 
3601 TACAGACAAG CTGTGACCGT CTCCGGGAGC TGCATGTGTC AGAGGTTTTC ACCGTCATCA 
3661 CCGAAACGCG CGAGACGAAA GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG 
3721 ATAATAATGG TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT 
3781 ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA 
3841 TAAATGCTTC AATAATATTG AAAAAGGAAG AGTATGAGTA TTCAACATTT CCGTGTCGCC 
3901 CTTATTCCCT TTTTTGCGGC ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG 
3961 AAAGTAAAAG ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC 
4021 AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT GATGAGCACT 
4081 TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTATTG ACGCCGGGCA AGAGCAACTC 
4141 GGTCGCCGCA TACACTATTC TCAGAATGAC TTGGTTGAGT ACTCACCAGT CACAGAAAAG 
4201 CATCTTACGG ATGGCATGAC AGTAAGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT 
4261 AACACTGCGG CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT 
4321 TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA GCTGAATGAA 
4381 GCCATACCAA ACGACGAGCG TGACACCACG ATGCCTGTAG CAATGGCAAC AACGTTGCGC 
4441 AAACTATTAA CTGGCGAACT ACTTACTCTA GCTTCCCGGC AACAATTAAT AGACTGGATG 
4501 GAGGCGGATA AAGTTGCAGG ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTGGTTTATT 
4561 GCTGATAAAT CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA 
4621 GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC AACTATGGAT 
4681 GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA TTAAGCATTG GTAACTGTCA 
4741 GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC TTCATTTTTA ATTTAAAAGG 
4801 ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA TCCCTTAACG TGAGTTTTCG 
4861 TTCCACTGAG CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT 
4921 CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT GGTTTGTTTG 
4981 CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG GCTTCAGCAG AGCGCAGATA 
5041 CCAAATACTG TTCTTCTAGT GTAGCCGTAG TTAGGCCACC ACTTCAAGAA CTCTGTAGCA 
5101 CCGCCTACAT ACCTCGCTCT GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG 
5161 TCGTGTCTTA CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 
5221 TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC CGAACTGAGA 
5281 TACCTACAGC GTGAGCTATG AGAAAGCGCC ACGCTTCCCG AAGGGAGAAA GGCGGACAGG 
5341 TATCCGGTAA GCGGCAGGGT CGGAACAGGA GAGCGCACGA GGGAGCTTCC AGGGGGAAAC 
5401 GCCTGGTATC TTTATAGTCC TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG 
5461 TGATGCTCGT CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG 
5521 TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGGCTCGAC AGATCT