| 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 |
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-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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