pAcGFP1-N1 encodes a green fluorescent protein (GFP) from Aequorea coerulescens (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). The MCS in pAcGFP1-N1 is between the immediate early promoter of CMV (PCMV IE) and the AcGFP1 coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of AcGFP1 if they are in the same reading frame as AcGFP1 and there are no intervening stop codons. SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3' end of the AcGFP1 mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor), consisting 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, allows stably transfected eukaryotic cells to be selected using G418. A bacterial promoter upstream of the gene expresses kanamycin resistance in E. coli. The pAcGFP1-N1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

載體應(yīng)用

Fusions to the N terminus of AcGFP1 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pAcGFP1-N1 so that it is in frame with the AcGFP1 coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (2). pAcGFP1-N1 can also be used simply to express AcGFP1 in a cell line of interest (e.g., as a transfection marker).

0001 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG 
0061 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT 
0121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA 
0181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC 
0241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA 
0301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC 
0361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG 
0421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG 
0481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT 
0541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA 
0601 CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG 
0661 GATCCACCGG TCATGGTGAG CAAGGGCGCC GAGCTGTTCA CCGGCATCGT GCCCATCCTG 
0721 ATCGAGCTGA ATGGCGATGT GAATGGCCAC AAGTTCAGCG TGAGCGGCGA GGGCGAGGGC 
0781 GATGCCACCT ACGGCAAGCT GACCCTGAAG TTCATCTGCA CCACCGGCAA GCTGCCTGTG 
0841 CCCTGGCCCA CCCTGGTGAC CACCCTGAGC TACGGCGTGC AGTGCTTCTC ACGCTACCCC 
0901 GATCACATGA AGCAGCACGA CTTCTTCAAG AGCGCCATGC CTGAGGGCTA CATCCAGGAG 
0961 CGCACCATCT TCTTCGAGGA TGACGGCAAC TACAAGTCGC GCGCCGAGGT GAAGTTCGAG 
1021 GGCGATACCC TGGTGAATCG CATCGAGCTG ACCGGCACCG ATTTCAAGGA GGATGGCAAC 
1081 ATCCTGGGCA ATAAGATGGA GTACAACTAC AACGCCCACA ATGTGTACAT CATGACCGAC 
1141 AAGGCCAAGA ATGGCATCAA GGTGAACTTC AAGATCCGCC ACAACATCGA GGATGGCAGC 
1201 GTGCAGCTGG CCGACCACTA CCAGCAGAAT ACCCCCATCG GCGATGGCCC TGTGCTGCTG 
1261 CCCGATAACC ACTACCTGTC CACCCAGAGC GCCCTGTCCA AGGACCCCAA CGAGAAGCGC 
1321 GATCACATGA TCTACTTCGG CTTCGTGACC GCCGCCGCCA TCACCCACGG CATGGATGAG 
1381 CTGTACAAGT GAGCGGCCGC GACTCTAGAT CATAATCAGC CATACCACAT TTGTAGAGGT 
1441 TTTACTTGCT TTAAAAAACC TCCCACACCT CCCCCTGAAC CTGAAACATA AAATGAATGC 
1501 AATTGTTGTT GTTAACTTGT TTATTGCAGC TTATAATGGT TACAAATAAA GCAATAGCAT 
1561 CACAAATTTC ACAAATAAAG CATTTTTTTC ACTGCATTCT AGTTGTGGTT TGTCCAAACT 
1621 CATCAATGTA TCTTAAGGCG TAAATTGTAA GCGTTAATAT TTTGTTAAAA TTCGCGTTAA 
1681 ATTTTTGTTA AATCAGCTCA TTTTTTAACC AATAGGCCGA AATCGGCAAA ATCCCTTATA 
1741 AATCAAAAGA ATAGACCGAG ATAGGGTTGA GTGTTGTTCC AGTTTGGAAC AAGAGTCCAC 
1801 TATTAAAGAA CGTGGACTCC AACGTCAAAG GGCGAAAAAC CGTCTATCAG GGCGATGGCC 
1861 CACTACGTGA ACCATCACCC TAATCAAGTT TTTTGGGGTC GAGGTGCCGT AAAGCACTAA 
1921 ATCGGAACCC TAAAGGGAGC CCCCGATTTA GAGCTTGACG GGGAAAGCCG GCGAACGTGG 
1981 CGAGAAAGGA AGGGAAGAAA GCGAAAGGAG CGGGCGCTAG GGCGCTGGCA AGTGTAGCGG 
2041 TCACGCTGCG CGTAACCACC ACACCCGCCG CGCTTAATGC GCCGCTACAG GGCGCGTCAG 
2101 GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT 
2161 CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA 
2221 GGAAGAGTCC TGAGGCGGAA AGAACCAGCT GTGGAATGTG TGTCAGTTAG GGTGTGGAAA 
2281 GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC 
2341 CAGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA 
2401 TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC CCGCCCCTAA CTCCGCCCAG 
2461 TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT ATTTATGCAG AGGCCGAGGC 
2521 CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG GCCTAGGCTT 
2581 TTGCAAAGAT CGATCAAGAG ACAGGATGAG GATCGTTTCG CATGATTGAA CAAGATGGAT 
2641 TGCACGCAGG TTCTCCGGCC GCTTGGGTGG AGAGGCTATT CGGCTATGAC TGGGCACAAC 
2701 AGACAATCGG CTGCTCTGAT GCCGCCGTGT TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC 
2761 TTTTTGTCAA GACCGACCTG TCCGGTGCCC TGAATGAACT GCAAGACGAG GCAGCGCGGC 
2821 TATCGTGGCT GGCCACGACG GGCGTTCCTT GCGCAGCTGT GCTCGACGTT GTCACTGAAG 
2881 CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG TGCCGGGGCA GGATCTCCTG TCATCTCACC 
2941 TTGCTCCTGC CGAGAAAGTA TCCATCATGG CTGATGCAAT GCGGCGGCTG CATACGCTTG 
3001 ATCCGGCTAC CTGCCCATTC GACCACCAAG CGAAACATCG CATCGAGCGA GCACGTACTC 
3061 GGATGGAAGC CGGTCTTGTC GATCAGGATG ATCTGGACGA AGAGCATCAG GGGCTCGCGC 
3121 CAGCCGAACT GTTCGCCAGG CTCAAGGCGA GCATGCCCGA CGGCGAGGAT CTCGTCGTGA 
3181 CCCATGGCGA TGCCTGCTTG CCGAATATCA TGGTGGAAAA TGGCCGCTTT TCTGGATTCA 
3241 TCGACTGTGG CCGGCTGGGT GTGGCGGACC GCTATCAGGA CATAGCGTTG GCTACCCGTG 
3301 ATATTGCTGA AGAGCTTGGC GGCGAATGGG CTGACCGCTT CCTCGTGCTT TACGGTATCG 
3361 CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT TGACGAGTTC TTCTGAGCGG 
3421 GACTCTGGGG TTCGAAATGA CCGACCAAGC GACGCCCAAC CTGCCATCAC GAGATTTCGA 
3481 TTCCACCGCC GCCTTCTATG AAAGGTTGGG CTTCGGAATC GTTTTCCGGG ACGCCGGCTG 
3541 GATGATCCTC CAGCGCGGGG ATCTCATGCT GGAGTTCTTC GCCCACCCTA GGGGGAGGCT 
3601 AACTGAAACA CGGAAGGAGA CAATACCGGA AGGAACCCGC GCTATGACGG CAATAAAAAG 
3661 ACAGAATAAA ACGCACGGTG TTGGGTCGTT TGTTCATAAA CGCGGGGTTC GGTCCCAGGG 
3721 CTGGCACTCT GTCGATACCC CACCGAGACC CCATTGGGGC CAATACGCCC GCGTTTCTTC 
3781 CTTTTCCCCA CCCCACCCCC CAAGTTCGGG TGAAGGCCCA GGGCTCGCAG CCAACGTCGG 
3841 GGCGGCAGGC CCTGCCATAG CCTCAGGTTA CTCATATATA CTTTAGATTG ATTTAAAACT 
3901 TCATTTTTAA TTTAAAAGGA TCTAGGTGAA GATCCTTTTT GATAATCTCA TGACCAAAAT 
3961 CCCTTAACGT GAGTTTTCGT TCCACTGAGC GTCAGACCCC GTAGAAAAGA TCAAAGGATC 
4021 TTCTTGAGAT CCTTTTTTTC TGCGCGTAAT CTGCTGCTTG CAAACAAAAA AACCACCGCT 
4081 ACCAGCGGTG GTTTGTTTGC CGGATCAAGA GCTACCAACT CTTTTTCCGA AGGTAACTGG 
4141 CTTCAGCAGA GCGCAGATAC CAAATACTGT CCTTCTAGTG TAGCCGTAGT TAGGCCACCA 
4201 CTTCAAGAAC TCTGTAGCAC CGCCTACATA CCTCGCTCTG CTAATCCTGT TACCAGTGGC 
4261 TGCTGCCAGT GGCGATAAGT CGTGTCTTAC CGGGTTGGAC TCAAGACGAT AGTTACCGGA 
4321 TAAGGCGCAG CGGTCGGGCT GAACGGGGGG TTCGTGCACA CAGCCCAGCT TGGAGCGAAC 
4381 GACCTACACC GAACTGAGAT ACCTACAGCG TGAGCTATGA GAAAGCGCCA CGCTTCCCGA 
4441 AGGGAGAAAG GCGGACAGGT ATCCGGTAAG CGGCAGGGTC GGAACAGGAG AGCGCACGAG 
4501 GGAGCTTCCA GGGGGAAACG CCTGGTATCT TTATAGTCCT GTCGGGTTTC GCCACCTCTG 
4561 ACTTGAGCGT CGATTTTTGT GATGCTCGTC AGGGGGGCGG AGCCTATGGA AAAACGCCAG 
4621 CAACGCGGCC TTTTTACGGT TCCTGGCCTT TTGCTGGCCT TTTGCTCACA TGTTCTTTCC 
4681 TGCGTTATCC CCTGATTCTG TGGATAACCG TATTACCGCC ATGCAT