pAcGFP1-Golgi

pAcGFP1-Golgi

pAcGFP1-Golgi载体基本信息

pAcGFP1-Golgi载体质粒图谱和多克隆位点信息

pAcGFP1-Golgi载体简介

pAcGFP1-Golgi Vector encodes a fusion protein consisting of the N-terminal 81 amino acids of the human beta 1,4-galactosyltransferase (GT; 1) directly upstream of the green fluorescent protein (GFP) from Aequorea coerulescens (AcGFP1). The human beta 1,4-galactosyltransferase sequence contains a membrane-anchoring signal peptide that targets the protein to the transmedial region of the Golgi aparatus (2–4). AcGFP1 (excitation maximum = 475 nm; emission maximum = 505 nm) 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. SV40 polyadenylation signals downstream of the protein coding region direct proper processing of the 3' end of the mRNA. The vector contains an SV40 origin for replication and a neomycin resistance (Neor) gene for selection (using G418) in eukaryotic cells (5). 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-Golgi Vector is designed for fluorescent labeling of the Golgi apparatus. pAcGFP1-Golgi can be introduced into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (5). pAcGFP1-Golgi is not intended as a cloning vector; however, the backbone does contain unique restriction sites upstream and downstream of the AcGFP1-Golgi sequence which permit its excision.

Propagation in E. coli

 Suitable host strains: Fusion-Blue competent cells (Cat. No. 636700), 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) in E. coli hosts.

 E. coli replication origin: pUC

 Copy number: ≈500

 Plasmid incompatibility group: pMB1/ColE1

pAcGFP1-Golgi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 GCTAGCATGA

      601 GGCTTCGGGA GCCGCTCCTG AGCGGCAGCG CCGCGATGCC AGGCGCGTCC CTACAGCGGG

      661 CCTGCCGCCT GCTCGTGGCC GTCTGCGCTC TGCACCTTGG CGTCACCCTC GTTTACTACC

      721 TGGCTGGCCG CGACCTGAGC CGCCTGCCCC AACTGGTCGG AGTCTCCACA CCGCTGCAGG

      781 GCGGCTCGAA CAGTGCCGCC GCCATCGGGC AGTCCTCCGG GGAGCTCCGG ACCGGAGGGG

      841 CCAAGGATCC ACCGGTCATG GTGAGCAAGG GCGCCGAGCT GTTCACCGGC ATCGTGCCCA

      901 TCCTGATCGA GCTGAATGGC GATGTGAATG GCCACAAGTT CAGCGTGAGC GGCGAGGGCG

      961 AGGGCGATGC CACCTACGGC AAGCTGACCC TGAAGTTCAT CTGCACCACC GGCAAGCTGC

     1021 CTGTGCCCTG GCCCACCCTG GTGACCACCC TGAGCTACGG CGTGCAGTGC TTCTCACGCT

     1081 ACCCCGATCA CATGAAGCAG CACGACTTCT TCAAGAGCGC CATGCCTGAG GGCTACATCC

     1141 AGGAGCGCAC CATCTTCTTC GAGGATGACG GCAACTACAA GTCGCGCGCC GAGGTGAAGT

     1201 TCGAGGGCGA TACCCTGGTG AATCGCATCG AGCTGACCGG CACCGATTTC AAGGAGGATG

     1261 GCAACATCCT GGGCAATAAG ATGGAGTACA ACTACAACGC CCACAATGTG TACATCATGA

     1321 CCGACAAGGC CAAGAATGGC ATCAAGGTGA ACTTCAAGAT CCGCCACAAC ATCGAGGATG

     1381 GCAGCGTGCA GCTGGCCGAC CACTACCAGC AGAATACCCC CATCGGCGAT GGCCCTGTGC

     1441 TGCTGCCCGA TAACCACTAC CTGTCCACCC AGAGCGCCCT GTCCAAGGAC CCCAACGAGA

     1501 AGCGCGATCA CATGATCTAC TTCGGCTTCG TGACCGCCGC CGCCATCACC CACGGCATGG

     1561 ATGAGCTGTA CAAGTGAGCG GCCGCGACTC TAGATCATAA TCAGCCATAC CACATTTGTA

     1621 GAGGTTTTAC TTGCTTTAAA AAACCTCCCA CACCTCCCCC TGAACCTGAA ACATAAAATG

     1681 AATGCAATTG TTGTTGTTAA CTTGTTTATT GCAGCTTATA ATGGTTACAA ATAAAGCAAT

     1741 AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC ATTCTAGTTG TGGTTTGTCC

     1801 AAACTCATCA ATGTATCTTA AGGCGTAAAT TGTAAGCGTT AATATTTTGT TAAAATTCGC

     1861 GTTAAATTTT TGTTAAATCA GCTCATTTTT TAACCAATAG GCCGAAATCG GCAAAATCCC

     1921 TTATAAATCA AAAGAATAGA CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG

     1981 TCCACTATTA AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA

     2041 TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC

     2101 ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT TGACGGGGAA AGCCGGCGAA

     2161 CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT

     2221 AGCGGTCACG CTGCGCGTAA CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC

     2281 GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTAT TTTTCTAAAT

     2341 ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG

     2401 AAAAAGGAAG AGTCCTGAGG CGGAAAGAAC CAGCTGTGGA ATGTGTGTCA GTTAGGGTGT

     2461 GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA

     2521 GCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT

     2581 CTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC CCATCCCGCC CCTAACTCCG

     2641 CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCC

     2701 GAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG GAGGCTTTTT TGGAGGCCTA

     2761 GGCTTTTGCA AAGATCGATC AAGAGACAGG ATGAGGATCG TTTCGCATGA TTGAACAAGA

     2821 TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG CTATTCGGCT ATGACTGGGC

     2881 ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC

     2941 GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT GAACTGCAAG ACGAGGCAGC

     3001 GCGGCTATCG TGGCTGGCCA CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC

     3061 TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG GGGCAGGATC TCCTGTCATC

     3121 TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT GCAATGCGGC GGCTGCATAC

     3181 GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA CATCGCATCG AGCGAGCACG

     3241 TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG GACGAAGAGC ATCAGGGGCT

     3301 CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GGCGAGCATG CCCGACGGCG AGGATCTCGT

     3361 CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG GAAAATGGCC GCTTTTCTGG

     3421 ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT CAGGACATAG CGTTGGCTAC

     3481 CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG TGCTTTACGG

     3541 TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC CTTCTTGACG AGTTCTTCTG

     3601 AGCGGGACTC TGGGGTTCGA AATGACCGAC CAAGCGACGC CCAACCTGCC ATCACGAGAT

     3661 TTCGATTCCA CCGCCGCCTT CTATGAAAGG TTGGGCTTCG GAATCGTTTT CCGGGACGCC

     3721 GGCTGGATGA TCCTCCAGCG CGGGGATCTC ATGCTGGAGT TCTTCGCCCA CCCTAGGGGG

     3781 AGGCTAACTG AAACACGGAA GGAGACAATA CCGGAAGGAA CCCGCGCTAT GACGGCAATA

     3841 AAAAGACAGA ATAAAACGCA CGGTGTTGGG TCGTTTGTTC ATAAACGCGG GGTTCGGTCC

     3901 CAGGGCTGGC ACTCTGTCGA TACCCCACCG AGACCCCATT GGGGCCAATA CGCCCGCGTT

     3961 TCTTCCTTTT CCCCACCCCA CCCCCCAAGT TCGGGTGAAG GCCCAGGGCT CGCAGCCAAC

     4021 GTCGGGGCGG CAGGCCCTGC CATAGCCTCA GGTTACTCAT ATATACTTTA GATTGATTTA

     4081 AAACTTCATT TTTAATTTAA AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC

     4141 AAAATCCCTT AACGTGAGTT TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA

     4201 GGATCTTCTT GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA

     4261 CCGCTACCAG CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA

     4321 ACTGGCTTCA GCAGAGCGCA GATACCAAAT ACTGTTCTTC TAGTGTAGCC GTAGTTAGGC

     4381 CACCACTTCA AGAACTCTGT AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA

     4441 GTGGCTGCTG CCAGTGGCGA TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA

     4501 CCGGATAAGG CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG

     4561 CGAACGACCT ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT

     4621 CCCGAAGGGA GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC

     4681 ACGAGGGAGC TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC

     4741 CTCTGACTTG AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC

     4801 GCCAGCAACG CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC

     4861 TTTCCTGCGT TATCCCCTGA TTCTGTGGAT AACCGTATTA CCGCCATGCA T

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