pAcGFP1-1

pAcGFP1-1 encodes the green fluorescent protein AcGFP1, a derivative of AcGFP from Aequorea coerulescens. AcGFP1 has been optimized for brighter fluorescence. (Excitation maximum = 475 nm; emission maximum = 505 nm.) The coding sequence of the AcGFP1 gene contains silent base changes, which correspond to human codon-usage preferences (1).

pAcGFP1-1 is a promoterless vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). Sequences upstream of AcGFP1 have been converted to a Kozak consensus translation initiation site (2) to enhance translation efficiency in eukaryotic cells. SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3' end of the AcGFP1 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.

载体应用

AcGFP1 can be used as an in vivo reporter of gene expression. Promoters should be cloned into the pAcGFP1-1 MCS upstream from the AcGFP1 coding sequence. Without addition of a functional promoter, this vector will not express AcGFP1. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard method. If required, stable transfectants can be selected using G418 (3).

0001 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG 
0061 ACGGTACCGC GGGCCCGGGA TCCACCGGTC GCCACCATGG TGAGCAAGGG CGCCGAGCTG 
0121 TTCACCGGCA TCGTGCCCAT CCTGATCGAG CTGAATGGCG ATGTGAATGG CCACAAGTTC 
0181 AGCGTGAGCG GCGAGGGCGA GGGCGATGCC ACCTACGGCA AGCTGACCCT GAAGTTCATC 
0241 TGCACCACCG GCAAGCTGCC TGTGCCCTGG CCCACCCTGG TGACCACCCT GAGCTACGGC 
0301 GTGCAGTGCT TCTCACGCTA CCCCGATCAC ATGAAGCAGC ACGACTTCTT CAAGAGCGCC 
0361 ATGCCTGAGG GCTACATCCA GGAGCGCACC ATCTTCTTCG AGGATGACGG CAACTACAAG 
0421 TCGCGCGCCG AGGTGAAGTT CGAGGGCGAT ACCCTGGTGA ATCGCATCGA GCTGACCGGC 
0481 ACCGATTTCA AGGAGGATGG CAACATCCTG GGCAATAAGA TGGAGTACAA CTACAACGCC 
0541 CACAATGTGT ACATCATGAC CGACAAGGCC AAGAATGGCA TCAAGGTGAA CTTCAAGATC 
0601 CGCCACAACA TCGAGGATGG CAGCGTGCAG CTGGCCGACC ACTACCAGCA GAATACCCCC 
0661 ATCGGCGATG GCCCTGTGCT GCTGCCCGAT AACCACTACC TGTCCACCCA GAGCGCCCTG 
0721 TCCAAGGACC CCAACGAGAA GCGCGATCAC ATGATCTACT TCGGCTTCGT GACCGCCGCC 
0781 GCCATCACCC ACGGCATGGA TGAGCTGTAC AAGTGAGCGG CCGCGACTCT AGATCATAAT 
0841 CAGCCATACC ACATTTGTAG AGGTTTTACT TGCTTTAAAA AACCTCCCAC ACCTCCCCCT 
0901 GAACCTGAAA CATAAAATGA ATGCAATTGT TGTTGTTAAC TTGTTTATTG CAGCTTATAA 
0961 TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT AAAGCATTTT TTTCACTGCA 
1021 TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAA GGCGTAAATT GTAAGCGTTA 
1081 ATATTTTGTT AAAATTCGCG TTAAATTTTT GTTAAATCAG CTCATTTTTT AACCAATAGG 
1141 CCGAAATCGG CAAAATCCCT TATAAATCAA AAGAATAGAC CGAGATAGGG TTGAGTGTTG 
1201 TTCCAGTTTG GAACAAGAGT CCACTATTAA AGAACGTGGA CTCCAACGTC AAAGGGCGAA 
1261 AAACCGTCTA TCAGGGCGAT GGCCCACTAC GTGAACCATC ACCCTAATCA AGTTTTTTGG 
1321 GGTCGAGGTG CCGTAAAGCA CTAAATCGGA ACCCTAAAGG GAGCCCCCGA TTTAGAGCTT 
1381 GACGGGGAAA GCCGGCGAAC GTGGCGAGAA AGGAAGGGAA GAAAGCGAAA GGAGCGGGCG 
1441 CTAGGGCGCT GGCAAGTGTA GCGGTCACGC TGCGCGTAAC CACCACACCC GCCGCGCTTA 
1501 ATGCGCCGCT ACAGGGCGCG TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA 
1561 TTTGTTTATT TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT 
1621 AAATGCTTCA ATAATATTGA AAAAGGAAGA GTCCTGAGGC GGAAAGAACC AGCTGTGGAA 
1681 TGTGTGTCAG TTAGGGTGTG GAAAGTCCCC AGGCTCCCCA GCAGGCAGAA GTATGCAAAG 
1741 CATGCATCTC AATTAGTCAG CAACCAGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG 
1801 AAGTATGCAA AGCATGCATC TCAATTAGTC AGCAACCATA GTCCCGCCCC TAACTCCGCC 
1861 CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT 
1921 TTTTATTTAT GCAGAGGCCG AGGCCGCCTC GGCCTCTGAG CTATTCCAGA AGTAGTGAGG 
1981 AGGCTTTTTT GGAGGCCTAG GCTTTTGCAA AGATCGATCA AGAGACAGGA TGAGGATCGT 
2041 TTCGCATGAT TGAACAAGAT GGATTGCACG CAGGTTCTCC GGCCGCTTGG GTGGAGAGGC 
2101 TATTCGGCTA TGACTGGGCA CAACAGACAA TCGGCTGCTC TGATGCCGCC GTGTTCCGGC 
2161 TGTCAGCGCA GGGGCGCCCG GTTCTTTTTG TCAAGACCGA CCTGTCCGGT GCCCTGAATG 
2221 AACTGCAAGA CGAGGCAGCG CGGCTATCGT GGCTGGCCAC GACGGGCGTT CCTTGCGCAG 
2281 CTGTGCTCGA CGTTGTCACT GAAGCGGGAA GGGACTGGCT GCTATTGGGC GAAGTGCCGG 
2341 GGCAGGATCT CCTGTCATCT CACCTTGCTC CTGCCGAGAA AGTATCCATC ATGGCTGATG 
2401 CAATGCGGCG GCTGCATACG CTTGATCCGG CTACCTGCCC ATTCGACCAC CAAGCGAAAC 
2461 ATCGCATCGA GCGAGCACGT ACTCGGATGG AAGCCGGTCT TGTCGATCAG GATGATCTGG 
2521 ACGAAGAGCA TCAGGGGCTC GCGCCAGCCG AACTGTTCGC CAGGCTCAAG GCGAGCATGC 
2581 CCGACGGCGA GGATCTCGTC GTGACCCATG GCGATGCCTG CTTGCCGAAT ATCATGGTGG 
2641 AAAATGGCCG CTTTTCTGGA TTCATCGACT GTGGCCGGCT GGGTGTGGCG GACCGCTATC 
2701 AGGACATAGC GTTGGCTACC CGTGATATTG CTGAAGAGCT TGGCGGCGAA TGGGCTGACC 
2761 GCTTCCTCGT GCTTTACGGT ATCGCCGCTC CCGATTCGCA GCGCATCGCC TTCTATCGCC 
2821 TTCTTGACGA GTTCTTCTGA GCGGGACTCT GGGGTTCGAA ATGACCGACC AAGCGACGCC 
2881 CAACCTGCCA TCACGAGATT TCGATTCCAC CGCCGCCTTC TATGAAAGGT TGGGCTTCGG 
2941 AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGC GGGGATCTCA TGCTGGAGTT 
3001 CTTCGCCCAC CCTAGGGGGA GGCTAACTGA AACACGGAAG GAGACAATAC CGGAAGGAAC 
3061 CCGCGCTATG ACGGCAATAA AAAGACAGAA TAAAACGCAC GGTGTTGGGT CGTTTGTTCA 
3121 TAAACGCGGG GTTCGGTCCC AGGGCTGGCA CTCTGTCGAT ACCCCACCGA GACCCCATTG 
3181 GGGCCAATAC GCCCGCGTTT CTTCCTTTTC CCCACCCCAC CCCCCAAGTT CGGGTGAAGG 
3241 CCCAGGGCTC GCAGCCAACG TCGGGGCGGC AGGCCCTGCC ATAGCCTCAG GTTACTCATA 
3301 TATACTTTAG ATTGATTTAA AACTTCATTT TTAATTTAAA AGGATCTAGG TGAAGATCCT 
3361 TTTTGATAAT CTCATGACCA AAATCCCTTA ACGTGAGTTT TCGTTCCACT GAGCGTCAGA 
3421 CCCCGTAGAA AAGATCAAAG GATCTTCTTG AGATCCTTTT TTTCTGCGCG TAATCTGCTG 
3481 CTTGCAAACA AAAAAACCAC CGCTACCAGC GGTGGTTTGT TTGCCGGATC AAGAGCTACC 
3541 AACTCTTTTT CCGAAGGTAA CTGGCTTCAG CAGAGCGCAG ATACCAAATA CTGTCCTTCT 
3601 AGTGTAGCCG TAGTTAGGCC ACCACTTCAA GAACTCTGTA GCACCGCCTA CATACCTCGC 
3661 TCTGCTAATC CTGTTACCAG TGGCTGCTGC CAGTGGCGAT AAGTCGTGTC TTACCGGGTT 
3721 GGACTCAAGA CGATAGTTAC CGGATAAGGC GCAGCGGTCG GGCTGAACGG GGGGTTCGTG 
3781 CACACAGCCC AGCTTGGAGC GAACGACCTA CACCGAACTG AGATACCTAC AGCGTGAGCT 
3841 ATGAGAAAGC GCCACGCTTC CCGAAGGGAG AAAGGCGGAC AGGTATCCGG TAAGCGGCAG 
3901 GGTCGGAACA GGAGAGCGCA CGAGGGAGCT TCCAGGGGGA AACGCCTGGT ATCTTTATAG 
3961 TCCTGTCGGG TTTCGCCACC TCTGACTTGA GCGTCGATTT TTGTGATGCT CGTCAGGGGG 
4021 GCGGAGCCTA TGGAAAAACG CCAGCAACGC GGCCTTTTTA CGGTTCCTGG CCTTTTGCTG 
4081 GCCTTTTGCT CACATGTTCT TTCCTGCGTT ATCCCCTGAT TCTGTGGATA ACCGTATTAC 
4141 CGCCATGCAT