pAcGFP1-F

价格:2500元

联系方式:I47-825O-882O

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载体基本信息

出品公司: Clontech
载体名称: pAcGFP1-F
质粒类型: 哺乳动物细胞表达载体;荧光报告载体
高拷贝/低拷贝: 高拷贝
克隆方法: 限制性内切酶,多克隆位点
启动子: CMV IE
载体大小: 4806 bp
5' 测序引物及序列: --
3' 测序引物及序列: --
载体标签: AcGFP1 (N-端)
载体抗性: 卡那霉素
筛选标记: 新霉素(Neomycin)
克隆菌株: DH5α, HB101
宿主细胞(系): 常规细胞系(293、CV-1、CHO等)
备注: 哺乳动物载体pAcGFP1-F组成型表达Ras-AcGFP融合蛋白;融合蛋白定位于细胞内膜上,荧光标记细胞膜。
产品目录号: 632511
稳定性: 稳表达 或 瞬表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

载体质粒图谱和多克隆位点信息

pAcGFP1-F载体图谱



pAcGFP1-F 载体特征

载体简介

pAcGFP1-F载体描述

Ras (1, 2) fused to the C-terminus of AcGFP1. Post-translation of this farnesylation signal targets AcGFP1-F to the inner leaflet of the plasma membrane. AcGFP1 is derived from Aequorea coerulescens. When AcGFP1-F is expressed in mammalian cell cultures, green fluorescent cells can be detected by either fluorescence microscopy or flow cytometry 12–16 hr after transfection (excitation maximum = 475 nm; emission maximum = 505 nm, respectively). The AcGFP1 coding sequence is human-codon-optimized for increased translation efficiency in mammalian cells (3). SV40 polyadenylation signals downstream of the AcGFP1-F gene direct proper processing of the 3’ end of the AcGFP1-F 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 this cassette expresses kanamycin resistance in E. coli. The pAcGFP1-F backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.


pAcGFP1-F is designed for use as a plasma membrane marker, as well as a cotransfection marker. Because it remains attached to the plasma membrane, it can be detected by fluorescence microscopy in permeabilized cells after ethanol fixation (4). The vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (5).

Propagation in E. coli
 Suitable host strains: DH5a, HB101, and other general purpose strains. Single-stranded DNA production
requires a host containing an F plasmid such as JM109 or XL1-Blue.
 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
 E. coli replication origin: pUC; copy number: high


References
1. Aronheim, A., et al. (1994) Cell 78:949–961.
2. Hancock, J. F., et al. (1991) EMBO J. 10:4033–4039.
3. Haas, J., et al. (1996) Curr. Biol. 6:315–324.
4. Jiang, W. & Hunter, T. (1998) BioTechniques 24:348–354.
5. Gorman, C. (1985) In DNA Cloning: A Practical Approach, Vol. II, Ed. Glover, D. M. (IRL Press, Oxford, UK) pp. 143–190.
Note: The attached sequence file has been compiled from information in the sequence databases, published literature, and other sources,
together with partial sequences obtained by Clontech. This vector has not been completely sequenced.


载体序列

LOCUS       pAcGFP1-F   4806 bp    DNA   circular             7-AUG-2009
COMMENT     Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT     Cat. No. 632511
FEATURES             Location/Qualifiers
     misc_feature    1345..1407
                     /label=c-Ha-Ras\farnesylation
     promoter        1..589
                     /label=CMV\IE\Promoter
     rep_origin      2518..2656
                     /label=SV40\ori
     CDS             2702..3496
                     /label=Kan(R)/Neo(R)
                     /note="Kanamycin/neomycin resistance gene Neomycin phosphotransferase coding sequences: start codon (ATG): 2629-2631; stop codon: 3421-3423; G to A mutation to remove Pst I site: 2811; C to A mutation to remove BssH II site: 3157"
     rep_origin      4081..4724
                     /label=pUC\ori
     rep_origin      complement(1722..2177)
                     /label=f1_ori
     CDS             613..1329
                     /label=AcGFP1
     polyA_signal    1625..1630
                     /label=SV40\early\polyA\signal
     polyA_signal    1654..1659
                     /label=SV40\early\polyA\signal
     polyA_signal    3732..3737
                     /label=HSV\TK\Poly\A\signal
     polyA_signal    3745..3750
                     /label=HSV\TK\Poly\A\signal
     misc_feature    606..616
                     /label=Kozak\consensus\translation\initiation\site
     misc_feature    2574..2580
                     /label=SV40\early\promoter\element
BASE COUNT     1156 a      1291 c      1259 g      1100 t 
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 gctagcgcta 
      601 ccggtcgcca ccatggtgag caagggcgcc gagctgttca ccggcatcgt gcccatcctg 
      661 atcgagctga atggcgatgt gaatggccac aagttcagcg tgagcggcga gggcgagggc 
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcctgtg 
      781 ccctggccca ccctggtgac caccctgagc tacggcgtgc agtgcttctc acgctacccc 
      841 gatcacatga agcagcacga cttcttcaag agcgccatgc ctgagggcta catccaggag 
      901 cgcaccatct tcttcgagga tgacggcaac tacaagtcgc gcgccgaggt gaagttcgag 
      961 ggcgataccc tggtgaatcg catcgagctg accggcaccg atttcaagga ggatggcaac 
     1021 atcctgggca ataagatgga gtacaactac aacgcccaca atgtgtacat catgaccgac 
     1081 aaggccaaga atggcatcaa ggtgaacttc aagatccgcc acaacatcga ggatggcagc 
     1141 gtgcagctgg ccgaccacta ccagcagaat acccccatcg gcgatggccc tgtgctgctg 
     1201 cccgataacc actacctgtc cacccagagc gccctgtcca aggaccccaa cgagaagcgc 
     1261 gatcacatga tctacttcgg cttcgtgacc gccgccgcca tcacccacgg catggatgag 
     1321 ctgtacaagt ccggactcag atctaagctg aaccctcctg atgagagtgg ccccggctgc 
     1381 atgagctgca agtgtgtgct ctcctgagga tccagatctc gagctcaagc ttcgaattct 
     1441 gcagtcgacg gtaccgcggg cccgggatcc accggatcta gataactgat cataatcagc 
     1501 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac 
     1561 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt 
     1621 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 
     1681 agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat 
     1741 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 
     1801 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 
     1861 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac 
     1921 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 
     1981 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 
     2041 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag 
     2101 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 
     2161 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 
     2221 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 
     2281 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg 
     2341 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg 
     2401 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt 
     2461 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc 
     2521 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt 
     2581 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc 
     2641 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg 
     2701 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt 
     2761 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc 
     2821 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact 
     2881 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt 
     2941 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca 
     3001 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat 
     3061 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg 
     3121 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga 
     3181 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga 
     3241 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa 
     3301 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga 
     3361 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt 
     3421 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct 
     3481 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac 
     3541 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc 
     3601 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc 
     3661 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc 
     3721 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa 
     3781 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc 
     3841 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca 
     3901 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata 
     3961 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 
     4021 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 
     4081 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 
     4141 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 
     4201 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 
     4261 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 
     4321 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 
     4381 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 
     4441 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 
     4501 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 
     4561 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 
     4621 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 
     4681 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 
     4741 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 
     4801 atgcat 
//

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