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产品资料

pECFP-N1

pECFP-N1
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  • 产品名称：pECFP-N1
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  • 产品展商：XYbscience
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简单介绍
pECFP-N1编码Aequorea维多利亚绿色荧光蛋白基因（GFP）的增强的青色荧光变体。ECFP基因含有6个氨基酸取代基。 Tyr-66对Trpsubstitution给出ECFP荧光激发（433nm处的主峰和453nm处的次峰）和与其他青色发射变体类似的发射（475nm处的主峰和501nm的次峰）。pECFP-N1其他五个取代（Phe-64至Leu; Ser-65至Thr; Asn-146至Ile; Met-153至Thr;和Val-163至Ala）增强了蛋白质的亮度和溶解度，主要是由于改进的蛋白质折叠性质和生色团形成的效率。
产品描述

pECFP-N1

  • 产品信息
  • 基本信息
  • 质粒简介
  • 质粒图谱
  • 质粒序列

产品信息

产品货号 产品名称 产品规格 优惠价
XY0143 pECFP-N1

5μg

¥请询价

使用说明

信裕质粒平台的各批次质粒菌株发货前均经过严格的多重验证，如存在质量问题，请在收到产品的三个月内通知我司。收到质粒后请短暂离心，加入20μl ddH2O溶解质粒，取2μl转化至对应感受态中，挑取单克隆重新提取质粒后使用。如需获取其他详细信息请登录我司官网查询。

基本信息

启动子: CMV promoter
复制子: pUC ori,f1 ori
终止子: SV40 poly(A) signal
质粒分类: 哺乳系列质粒；哺乳荧光质粒；哺乳青色质粒
质粒大小: 4733bp
质粒标签: C-ECFP
原核抗性: 卡那霉素Kan（50μg/ml）
筛选标记: 新霉素Neo/G418
克隆菌株: DH5α等大肠杆菌
培养条件: 37℃，有氧 LB
表达宿主: 293T等哺乳细胞
培养条件: 37℃，5%CO2
诱导方式: 无须诱导，瞬时表达
5'测序引物: CMV-F（CGCAAATGGGCGGTAGGCGTG）
3'测序引物: Sv40-polyA-R（GAAATTTGTGATGCTATTGC）

质粒简介

        pECFP-N1编码Aequorea维多利亚绿色荧光蛋白基因（GFP）的增强的青色荧光变体。ECFP基因含有6个氨基酸取代基。 Tyr-66对Trpsubstitution给出ECFP荧光激发（433nm处的主峰和453nm处的次峰）和与其他青色发射变体类似的发射（475nm处的主峰和501nm的次峰）。其他五个取代（Phe-64至Leu; Ser-65至Thr; Asn-146至Ile; Met-153至Thr;和Val-163至Ala）增强了蛋白质的亮度和溶解度，主要是由于改进的蛋白质折叠性质和生色团形成的效率。除了发色团突变外，ECFP含有> 190个沉默突变，创造出几乎完全是优选人类密码子的开放阅读框架。此外，ECFP侧翼的上游序列已转化为Kozak一致翻译起始位点。这些变化增加了ECFP mRNA的翻译效率，从而增加了哺乳动物和植物细胞中ECFP的表达。     
       pECFP-N1 encodes an enhanced cyan fluorescent variant of the Aequorea victoria green fluorescent protein gene (GFP). The ECFP gene contains six amino acid substitutions. The Tyr-66 to Trp substitution gives ECFP fluorescence excitation (major peak at 433 nm and a minor peak at 453 nm) and emission (major peak at 475 nm and a minor peak at 501 nm) similar to other cyan emission variants (1, 2). The other five substitutions (Phe-64 to Leu; Ser-65 to Thr; Asn-146 to Ile; Met-153 to Thr; and Val-163 to Ala) enhance the brightness and solubility of the protein, primarily due to improved protein folding properties and efficiency of chromophore formation (1, 3, 4). In addition to the chromophore mutations, ECFP contains >190 silent mutations that create an open reading frame comprised almost entirely of preferred human codons (5, 6). Furthermore, upstream sequences flanking ECFP have been converted to a Kozak consensus translation initiation site (7). These changes increase the translational efficiency of the ECFP mRNA and consequently the expression of ECFP in mammalian and plant cells.
       The vector contains an SV40 origin of replication and a neomycin resistance (Neor ) gene for selection (using G418) 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. The MCS in pECFP-N1 is between the immediate early promoter of CMV (PCMV IE) and the ECFP coding sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of ECFP if they are in the same reading frame as ECFP and there are no intervening stop codons. The inserted gene should include an initiating ATG codon. ECFP fusion proteins retain the fluorescent properties of the native protein in vivo.
       The recombinant ECFP vector can be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (8). pECFP-N1 can also be used simply to express ECFP in a cell line of interest (e.g., as a transfection marker). ECFP can be used for double-labeling experiments together with EYFP using standard fluorescence microscopy and the appropriate filter sets.

质粒图谱



质粒序列

LOCUS       Exported                4733 bp ds-DNA    circular SYN 19-10-2015
KEYWORDS    Untitled
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4733)
  AUTHORS   admin
  TITLE     Direct Submission
  JOURNAL   Exported 2015-10-19  from MiaoLingPlasmid
            http://www.miaolingbio.com
FEATURES             Location/Qualifiers
     source          1..4733
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        61..364
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    591..671
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             679..1398
                     /codon_start=1
                     /product="enhanced CFP"
                     /note="ECFP"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    1521..1642
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1649..2104)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2131..2235
                     /gene="bla"
                     /note="AmpR promoter"
     promoter        2237..2594
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      2445..2580
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             2629..3423
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3655..3702
                     /note="HSV TK poly(A) signal"
                     /note="herpesvirus thymidine kinase polyadenylation signal"
     rep_origin      4031..4619
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
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 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      721 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      781 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      841 cccgtgccct ggcccaccct cgtgaccacc ctgacctggg gcgtgcagtg cttcagccgc
      901 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      961 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     1021 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     1081 ggcaacatcc tggggcacaa gctggagtac aactacatca gccacaacgt ctatatcacc
     1141 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
     1201 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     1261 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
     1321 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     1381 gacgagctgt acaagtaaag cggccgcgac tctagatcat aatcagccat accacatttg
     1441 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     1501 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     1561 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     1621 ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc
     1681 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     1741 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     1801 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     1861 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     1921 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     1981 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     2041 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     2101 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     2161 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2221 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     2281 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2341 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     2401 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     2461 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     2521 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     2581 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     2641 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     2701 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     2761 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
     2821 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     2881 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     2941 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     3001 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     3061 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     3121 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
     3181 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     3241 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     3301 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     3361 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
     3421 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
     3481 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     3541 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg
     3601 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa
     3661 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt
     3721 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg
     3781 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca
     3841 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt
     3901 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     3961 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4021 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4081 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4141 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     4201 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4261 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4321 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4381 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4441 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4501 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4561 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4621 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4681 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cat
//
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