pAcGFP1-Mito

pAcGFP1-Mito

pAcGFP1-Mito载体基本信息

pAcGFP1-Mito载体质粒图谱和多克隆位点信息

pAcGFP1-Mito载体简介

pAcGFP1-Mito encodes a fusion of a mitochondrial targeting sequence derived from the precursor of subunit VIII of human cytochrome C oxidase (1, 2) and the green fluorescent protein (GFP) from Aequorea coerulescens (AcGFP1; excitation maximum = 475 nm; emission maximum = 505 nm). The mitochondrial targeting sequence is fused to the N-terminus of AcGFP1. AcGFP1 contains silent mutations that create an open reading frame comprised almost entirely of optimized human codons. These changes increase the translational efficiency of the AcGFP1 mRNA and, consequently, the expression of AcGFP1 in mammalian and plant cells. The vector contains an SV40 origin for replication and a neomycin resistance (Neor) gene for selection (using G418) in eukaryotic cells (3). A bacterial promoter (P) upstream of Neor expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

pAcGFP1-Mito is designed for fluorescent labeling of mitochondria. The fluorescence from pAcGFP1-Mito expression can be observed within the mitochondrial matrix inside the inner membrane. pAcGFP1-Mito can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (3). pAcGFP1- Mito is not intended as a cloning vector; however, the backbone does contain unique restriction sites upstream and downstream of the AcGFP1-Mito sequence which permit excision of the AcGFP1-Mito sequence.

Propagation in E. coli

  Suitable host strains: DH5α, HB101, and other general purpose strains. Single-stranded DNA production requires a host such as JM109 or XL1-Blue that contains an F plasmid .

  Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) to E. coli hosts.

  E. coli replication origin: pUC

  Copy number: ≈500

  Plasmid incompatibility group: pMB1/ColE1

pAcGFP1-Mito载体序列

ORIGIN

        1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG

       61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT

      121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA

      181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC

      241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA

      301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC

      361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG

      421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG

      481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT

      541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCATGT

      601 CCGTCCTGAC GCCGCTGCTG CTGCGGGGCT TGACAGGCTC GGCCCGGCGG CTCCCAGTGC

      661 CGCGCGCCAA GATCCATTCG TTGGGGGATC CACCGGTCAT GGTGAGCAAG GGCGCCGAGC

      721 TGTTCACCGG CATCGTGCCC ATCCTGATCG AGCTGAATGG CGATGTGAAT GGCCACAAGT

      781 TCAGCGTGAG CGGCGAGGGC GAGGGCGATG CCACCTACGG CAAGCTGACC CTGAAGTTCA

      841 TCTGCACCAC CGGCAAGCTG CCTGTGCCCT GGCCCACCCT GGTGACCACC CTGAGCTACG

      901 GCGTGCAGTG CTTCTCACGC TACCCCGATC ACATGAAGCA GCACGACTTC TTCAAGAGCG

      961 CCATGCCTGA GGGCTACATC CAGGAGCGCA CCATCTTCTT CGAGGATGAC GGCAACTACA

     1021 AGTCGCGCGC CGAGGTGAAG TTCGAGGGCG ATACCCTGGT GAATCGCATC GAGCTGACCG

     1081 GCACCGATTT CAAGGAGGAT GGCAACATCC TGGGCAATAA GATGGAGTAC AACTACAACG

     1141 CCCACAATGT GTACATCATG ACCGACAAGG CCAAGAATGG CATCAAGGTG AACTTCAAGA

     1201 TCCGCCACAA CATCGAGGAT GGCAGCGTGC AGCTGGCCGA CCACTACCAG CAGAATACCC

     1261 CCATCGGCGA TGGCCCTGTG CTGCTGCCCG ATAACCACTA CCTGTCCACC CAGAGCGCCC

     1321 TGTCCAAGGA CCCCAACGAG AAGCGCGATC ACATGATCTA CTTCGGCTTC GTGACCGCCG

     1381 CCGCCATCAC CCACGGCATG GATGAGCTGT ACAAGTGAGC GGCCGCGACT CTAGATCATA

     1441 ATCAGCCATA CCACATTTGT AGAGGTTTTA CTTGCTTTAA AAAACCTCCC ACACCTCCCC

     1501 CTGAACCTGA AACATAAAAT GAATGCAATT GTTGTTGTTA ACTTGTTTAT TGCAGCTTAT

     1561 AATGGTTACA AATAAAGCAA TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG

     1621 CATTCTAGTT GTGGTTTGTC CAAACTCATC AATGTATCTT AAGGCGTAAA TTGTAAGCGT

     1681 TAATATTTTG TTAAAATTCG CGTTAAATTT TTGTTAAATC AGCTCATTTT TTAACCAATA

     1741 GGCCGAAATC GGCAAAATCC CTTATAAATC AAAAGAATAG ACCGAGATAG GGTTGAGTGT

     1801 TGTTCCAGTT TGGAACAAGA GTCCACTATT AAAGAACGTG GACTCCAACG TCAAAGGGCG

     1861 AAAAACCGTC TATCAGGGCG ATGGCCCACT ACGTGAACCA TCACCCTAAT CAAGTTTTTT

     1921 GGGGTCGAGG TGCCGTAAAG CACTAAATCG GAACCCTAAA GGGAGCCCCC GATTTAGAGC

     1981 TTGACGGGGA AAGCCGGCGA ACGTGGCGAG AAAGGAAGGG AAGAAAGCGA AAGGAGCGGG

     2041 CGCTAGGGCG CTGGCAAGTG TAGCGGTCAC GCTGCGCGTA ACCACCACAC CCGCCGCGCT

     2101 TAATGCGCCG CTACAGGGCG CGTCAGGTGG CACTTTTCGG GGAAATGTGC GCGGAACCCC

     2161 TATTTGTTTA TTTTTCTAAA TACATTCAAA TATGTATCCG CTCATGAGAC AATAACCCTG

     2221 ATAAATGCTT CAATAATATT GAAAAAGGAA GAGTCCTGAG GCGGAAAGAA CCAGCTGTGG

     2281 AATGTGTGTC AGTTAGGGTG TGGAAAGTCC CCAGGCTCCC CAGCAGGCAG AAGTATGCAA

     2341 AGCATGCATC TCAATTAGTC AGCAACCAGG TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC

     2401 AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA TAGTCCCGCC CCTAACTCCG

     2461 CCCATCCCGC CCCTAACTCC GCCCAGTTCC GCCCATTCTC CGCCCCATGG CTGACTAATT

     2521 TTTTTTATTT ATGCAGAGGC CGAGGCCGCC TCGGCCTCTG AGCTATTCCA GAAGTAGTGA

     2581 GGAGGCTTTT TTGGAGGCCT AGGCTTTTGC AAAGATCGAT CAAGAGACAG GATGAGGATC

     2641 GTTTCGCATG ATTGAACAAG ATGGATTGCA CGCAGGTTCT CCGGCCGCTT GGGTGGAGAG

     2701 GCTATTCGGC TATGACTGGG CACAACAGAC AATCGGCTGC TCTGATGCCG CCGTGTTCCG

     2761 GCTGTCAGCG CAGGGGCGCC CGGTTCTTTT TGTCAAGACC GACCTGTCCG GTGCCCTGAA

     2821 TGAACTGCAA GACGAGGCAG CGCGGCTATC GTGGCTGGCC ACGACGGGCG TTCCTTGCGC

     2881 AGCTGTGCTC GACGTTGTCA CTGAAGCGGG AAGGGACTGG CTGCTATTGG GCGAAGTGCC

     2941 GGGGCAGGAT CTCCTGTCAT CTCACCTTGC TCCTGCCGAG AAAGTATCCA TCATGGCTGA

     3001 TGCAATGCGG CGGCTGCATA CGCTTGATCC GGCTACCTGC CCATTCGACC ACCAAGCGAA

     3061 ACATCGCATC GAGCGAGCAC GTACTCGGAT GGAAGCCGGT CTTGTCGATC AGGATGATCT

     3121 GGACGAAGAG CATCAGGGGC TCGCGCCAGC CGAACTGTTC GCCAGGCTCA AGGCGAGCAT

     3181 GCCCGACGGC GAGGATCTCG TCGTGACCCA TGGCGATGCC TGCTTGCCGA ATATCATGGT

     3241 GGAAAATGGC CGCTTTTCTG GATTCATCGA CTGTGGCCGG CTGGGTGTGG CGGACCGCTA

     3301 TCAGGACATA GCGTTGGCTA CCCGTGATAT TGCTGAAGAG CTTGGCGGCG AATGGGCTGA

     3361 CCGCTTCCTC GTGCTTTACG GTATCGCCGC TCCCGATTCG CAGCGCATCG CCTTCTATCG

     3421 CCTTCTTGAC GAGTTCTTCT GAGCGGGACT CTGGGGTTCG AAATGACCGA CCAAGCGACG

     3481 CCCAACCTGC CATCACGAGA TTTCGATTCC ACCGCCGCCT TCTATGAAAG GTTGGGCTTC

     3541 GGAATCGTTT TCCGGGACGC CGGCTGGATG ATCCTCCAGC GCGGGGATCT CATGCTGGAG

     3601 TTCTTCGCCC ACCCTAGGGG GAGGCTAACT GAAACACGGA AGGAGACAAT ACCGGAAGGA

     3661 ACCCGCGCTA TGACGGCAAT AAAAAGACAG AATAAAACGC ACGGTGTTGG GTCGTTTGTT

     3721 CATAAACGCG GGGTTCGGTC CCAGGGCTGG CACTCTGTCG ATACCCCACC GAGACCCCAT

     3781 TGGGGCCAAT ACGCCCGCGT TTCTTCCTTT TCCCCACCCC ACCCCCCAAG TTCGGGTGAA

     3841 GGCCCAGGGC TCGCAGCCAA CGTCGGGGCG GCAGGCCCTG CCATAGCCTC AGGTTACTCA

     3901 TATATACTTT AGATTGATTT AAAACTTCAT TTTTAATTTA AAAGGATCTA GGTGAAGATC

     3961 CTTTTTGATA ATCTCATGAC CAAAATCCCT TAACGTGAGT TTTCGTTCCA CTGAGCGTCA

     4021 GACCCCGTAG AAAAGATCAA AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC

     4081 TGCTTGCAAA CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA

     4141 CCAACTCTTT TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA TACTGTTCTT

     4201 CTAGTGTAGC CGTAGTTAGG CCACCACTTC AAGAACTCTG TAGCACCGCC TACATACCTC

     4261 GCTCTGCTAA TCCTGTTACC AGTGGCTGCT GCCAGTGGCG ATAAGTCGTG TCTTACCGGG

     4321 TTGGACTCAA GACGATAGTT ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG

     4381 TGCACACAGC CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG

     4441 CTATGAGAAA GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC GGTAAGCGGC

     4501 AGGGTCGGAA CAGGAGAGCG CACGAGGGAG CTTCCAGGGG GAAACGCCTG GTATCTTTAT

     4561 AGTCCTGTCG GGTTTCGCCA CCTCTGACTT GAGCGTCGAT TTTTGTGATG CTCGTCAGGG

     4621 GGGCGGAGCC TATGGAAAAA CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC

     4681 TGGCCTTTTG CTCACATGTT CTTTCCTGCG TTATCCCCTG ATTCTGTGGA TAACCGTATT

     4741 ACCGCCATGC AT

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