| 出品公司: | Clontech |
|---|---|
| 载体名称: | pAcGFP1-Mem |
| 质粒类型: | 哺乳动物细胞表达载体；荧光报告载体；亚细胞定位载体 |
| 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: | 4.8 kb |
| 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-Mem组成型表达AcGFP，定位于细胞膜。 |
| 产品目录号: | 632491 |
| 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 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
pAcGFP1-Mem载体描述
pAcGFP1-Mem encodes a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin, also called GAP-43 (1), and the Aequorea coerulescens green fluorescent protein AcGFP1. (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 (2). The neuromodulin fragment contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets AcGFP1 to cellular membranes, and the plasma membrane in particular. Expression of AcGFP1-Mem is driven by the immediate early promoter of CMV (PCMV IE). The vector contains an SV40 origin of replication and a neomycin resistance (Neor) gene for selection in mammalian cells. A bacterial promoter upstream of this cassette (P) expresses kanamycin resistance in E. coli. The vector backbone also provides a pUC19 origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
pAcGFP1-Mem can be transfected into mammalian cells using any standard method. If required, stable transformants can be selected using G418 (3). Expression of AcGFP1-Mem in mammalian cells results in strong labeling of the plasma membrane and allows easy tracking of individual cells in a population. This membrane labeling also permits the study of fine cellular processes such as neuronal axons (4), leading edges of migrating cells, filopodia, or microvilli on cell surfaces. pAc- GFP1-Mem cannot be used as an exclusive plasma membrane marker because it also partially labels intracellular membranes.
Propagation in E. coli
Suitable host strains: DH5α, HB101, and other general-purpose strains. Single-stranded DNA production requires
a host containing an F plasmid such as JM101 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. Skene, J. H. P. & Virag, I. (1989) J. Cell. Biol. 108:613–625.
2. Haas, J., et al. (1996) Curr. Biol. 6:315–324.
3. Gorman, C. (1985) In DNA cloning: a practical approach, vol. II. Ed. D. M. Glover. (IRL Press, Oxford, U.K.), pp. 143–190.
4. Moriyoshi, K., et al. (1996) Neuron 16:255–260.
载体序列
LOCUS pAcGFP1-Mem_Hyg 5846 bp DNA circular 10-NOV-2009
COMMENT Created by Clontech Laboratories Inc. http://www.clontech.com
COMMENT Cat. No. 632491
FEATURES Location/Qualifiers
CDS 679..738
/label=Membrane
/note="Neuromodulin GAP-43"
CDS 739..1449
/label=AcGFP1
promoter 1..589
/label=CMV\IE\Promoter
rep_origin 5121..5764
/label=pUC\origin
rep_origin complement(1703..2158)
/label=f1\origin
rep_origin 2499..2637
/label=SV40\origin
CDS 4098..4958
/label=Amp(R)
CDS 2656..3681
/label=Hyg(R)
polyA_signal 1606..1611
/label=SV40\early\polyA\signal
polyA_signal 1635..1640
/label=SV40\early\polyA\signal
polyA_signal 3829..3834
/label=SV40\early\polyA\signal
polyA_signal 3858..3863
/label=SV40\early\polyA\signal
misc_feature 2555..2561
/label=SV40\early\Promoter
promoter 4028..4056
/label=Amp(R)\Promoter
BASE COUNT 1484 a 1487 c 1484 g 1391 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 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg
661 gatccaccgg tcgccaccat gctgtgctgt atgagaagaa ccaaacaggt tgaaaagaat
721 gatgaggacc aaaagatcag caagggcgcc gagctgttca ccggcatcgt gcccatcctg
781 atcgagctga atggcgatgt gaatggccac aagttcagcg tgagcggcga gggcgagggc
841 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcctgtg
901 ccctggccca ccctggtgac caccctgagc tacggcgtgc agtgcttctc acgctacccc
961 gatcacatga agcagcacga cttcttcaag agcgccatgc ctgagggcta catccaggag
1021 cgcaccatct tcttcgagga tgacggcaac tacaagtcgc gcgccgaggt gaagttcgag
1081 ggcgataccc tggtgaatcg catcgagctg accggcaccg atttcaagga ggatggcaac
1141 atcctgggca ataagatgga gtacaactac aacgcccaca atgtgtacat catgaccgac
1201 aaggccaaga atggcatcaa ggtgaacttc aagatccgcc acaacatcga ggatggcagc
1261 gtgcagctgg ccgaccacta ccagcagaat acccccatcg gcgatggccc tgtgctgctg
1321 cccgataacc actacctgtc cacccagagc gccctgtcca aggaccccaa cgagaagcgc
1381 gatcacatga tctacttcgg cttcgtgacc gccgccgcca tcacccacgg catggatgag
1441 ctgtacaagt gaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg
1501 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
1561 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
1621 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
1681 tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta
1741 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
1801 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
1861 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
1921 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
1981 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
2041 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
2101 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca
2161 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
2221 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
2281 aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa
2341 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
2401 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
2461 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
2521 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
2581 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
2641 tttgcaaaga tcgatatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
2701 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
2761 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
2821 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
2881 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
2941 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
3001 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
3061 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
3121 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
3181 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
3241 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
3301 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
3361 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
3421 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
3481 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
3541 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
3601 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
3661 cgtccgaggg caaaggaata gagttctaga ggatcataat cagccatacc acatttgtag
3721 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
3781 atgcaattgt tgttgttaac ttgtttattg cagcttgtaa tggttacaaa taaagcaata
3841 gcatcacaaa tttcacaaat aaagcatttt tttcactgcc tcgcgatcca caaccatggc
3901 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
3961 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
4021 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
4081 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
4141 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
4201 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
4261 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
4321 gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact
4381 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
4441 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
4501 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
4561 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
4621 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
4681 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
4741 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
4801 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
4861 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
4921 tcgctgagat aggtgcctca ctgattaagc attggtaagg cgcgccaggc cctgccatag
4981 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
5041 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
5101 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
5161 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
5221 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
5281 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
5341 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
5401 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
5461 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
5521 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
5581 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
5641 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
5701 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
5761 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
5821 tggataaccg tattaccgcc atgcat
//
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