pFC32K Nluc-CMV-neo-Flexi载体

NanoLuc萤光素酶融合表达载体pFC32K Nluc-CMV-neo-Flexi更小（19.1kDa）、更亮（比萤火虫和海肾萤光素酶亮度高 100 倍）的 NanoLuc萤光素酶（Nluc）非常适用于蛋白融合应用。NanoLuc 融合蛋白应用广泛：作为蛋白稳定性报告基因、生物发光法细胞成像（BLI）探针或作为供体信号用于生物发光共振能量转移（BRET）研究蛋白: 蛋白或蛋白: 小分子相互作用研究。NanoLuc 蛋白融合载体可方便的 N- 或 C-端融合 Nluc 和您感兴趣蛋白的融合蛋白；有两类形式载体可供选择：pNLF 载体系列：产生 N- 或 C- 端融合全长 Nluc 的融合蛋白或应用传统的克隆方法（多克隆位点）将分泌型 Nluc 克隆到感兴趣的蛋白 N 端。 pF 载体系列：使用 Flexi Vector Cloning System 产生 N- 端或 C- 端融合蛋白；Flexi Vector Cloning System 是一种直接克隆方法，基于两种稀有酶切位点 SgfI 和 PmeI，可以在 Flexi 载体之间转换蛋白编码序列，快速、GX和高保真。特点 - 优点 轻松定量蛋白丰度变化：使用 Nano-Glo Luciferase Assay System 进行单一试剂加样，来定量 NanoLuc 融合蛋白的信号，进而检测细胞内蛋白水平。 生物相关性更高：NanoLuc 报告基因光信号更强，内源性表达水平的 NanoLuc 融合蛋白即可被检测到，可避免过度表达造成的假象。 使蛋白的细胞内动态变化可视化：NanoLuc 报告基因光信号更强，缩短了成像曝光时间，无需反复激发样本，从而降低了激发造成的细胞毒性反应。 改进 BRET 研究技术：NanoLuc 拥有更强的光信号，蓝移的发射光谱，因而与荧光受体的光谱交叉更少，信噪比更佳，动态范围更宽。 灵活的克隆选择：可使用传统克隆方法或 Flexi 克隆系统轻松将 NanoLuc 与目标蛋白的 C 端或 N 端融合。 可实现从瞬时转染到稳定转染的轻松过渡：所有载体都含有用于建立稳定细胞系的哺乳动物筛选标志物。应用 化合物筛选 蛋白: 蛋白相互作用 蛋白: 小分子相互作用 生物发光成像 蛋白稳定性研究

LOCUS       KF811456                5119 bp    DNA     circular SYN 03-FEB-2014DEFINITION  NLuc CMV-Neo Flexi Vector pFC32K, complete sequence.ACCESSION   KF811456VERSION     KF811456.1  GI:576890565KEYWORDS    .SOURCE      NLuc CMV-Neo Flexi Vector pFC32K  ORGANISM  NLuc CMV-Neo Flexi Vector pFC32K            other sequences; artificial sequences; vectors.REFERENCE   1  (bases 1 to 5119)  AUTHORS   McNamara,B., Kopish,K. and Robers,M.  TITLE     Direct Submission  JOURNAL   Submitted (04-NOV-2013) Scientific Communications, Promega            Corporation, 5500 E. Cheryl Parkway, Madison, WI 53711, USAFEATURES             Location/Qualifiers     source          1..5119                     /organism="NLuc CMV-Neo Flexi Vector pFC32K"                     /mol_type="other DNA"                     /db_xref="taxon:1454399"     regulatory      1..742                     /regulatory_class="promoter"                     /note="CMV immediate early enhancer/promoter"     intron          857..989                     /note="chimeric"     regulatory      1033..1052                     /regulatory_class="promoter"                     /note="T7 promoter"     misc_feature    1056..1063                     /note="used in Flexi vector cloning; SgfI site"     CDS             1087..1422                     /codon_start=1                     /transl_table=11                     /product="barnase"                     /protein_id="AHH41355.1"                     /db_xref="GI:576890567"                     /translation="MAQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLA                     DVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTD                     HYQTFTKIR"     misc_feature    1442..1447                     /note="EcoICRI site"     misc_feature    1447..1461                     /note="linker, C-terminal NanoLuc"     CDS             <1462..1974                     /note="C-terminus"                     /codon_start=1                     /transl_table=11                     /product="NanoLuc luciferase"                     /protein_id="AHH41356.1"                     /db_xref="GI:576890568"                     /translation="VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDG                     VTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT                     GWRLCERILA"     regulatory      2108..2329                     /regulatory_class="polyA_signal_sequence"                     /note="SV40 late polyA signal"     regulatory      2428..2840                     /regulatory_class="promoter"                     /note="SV40 early enhancer/promoter"     regulatory      2854..2920                     /regulatory_class="promoter"                     /note="Em7 bacterial promoter"     gene            2934..3728                     /gene="neoR/kanR"     CDS             2934..3728                     /gene="neoR/kanR"                     /function="resistance to neomycin and kanamycin"                     /codon_start=1                     /transl_table=11                     /product="neomycin phosphotransferase"                     /protein_id="AHH41354.1"                     /db_xref="GI:576890566"                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR                     PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL                     LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY                     QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"     regulatory      3792..3840                     /regulatory_class="polyA_signal_sequence"                     /note="synthetic polyA signal"     rep_origin      4076..4112                     /note="ColE1-derived plasmid replication origin"ORIGIN              1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc      361 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc      661 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc      721 tggtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat      781 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc      841 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa      901 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac      961 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta     1021 aggctagagt attaatacga ctcactatag ggctagcgat cgccatggaa taagtaagga     1081 atccacatgg cacaggttat caacacgttt gacggggttg cggattatct tcagacatat     1141 cataagctac ctgataatta cattacaaaa tcagaagcac aagccctcgg ctgggtggca     1201 tcaaaaggga accttgcaga cgtcgctccg gggaaaagca tcggcggaga catcttctca     1261 aacagggaag gcaaactccc gggcaaaagc ggacgaacat ggcgtgaagc ggatattaac     1321 tatacatcag gcttcagaaa ttcagaccgg attctttact caagcgactg gctgatttac     1381 aaaacaacgg accattatca gacctttaca aaaatcagat aatgtttaat gaccccgtgt     1441 cgagctctcg gctcgagcgg cgtcttcaca ctcgaagatt tcgttgggga ctggcgacag     1501 acagccggct acaacctgga ccaagtcctt gaacagggag gtgtgtccag tttgtttcag     1561 aatctcgggg tgtccgtaac tccgatccaa aggattgtcc tgagcggtga aaatgggctg     1621 aagatcgaca tccatgtcat catcccgtat gaaggtctga gcggcgacca aatgggccag     1681 atcgaaaaaa tttttaaggt ggtgtaccct gtggatgatc atcactttaa ggtgatcctg     1741 cactatggca cactggtaat cgacggggtt acgccgaaca tgatcgacta tttcggacgg     1801 ccgtatgaag gcatcgccgt gttcgacggc aaaaagatca ctgtaacagg gaccctgtgg     1861 aacggcaaca aaattatcga cgagcgcctg atcaaccccg acggctccct gctgttccga     1921 gtaaccatca acggagtgac cggctggcgg ctgtgcgaac gcattctggc gtaaggccgc     1981 gactctagag tcgacctgca ggcatgcaag ctgatccggc tgctaacaaa gcccgaaagg     2041 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcggccg     2101 cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag     2161 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata     2221 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg     2281 gagatgtggg aggttttttt aagcaagtaa aacctctaca aatgtggtaa aatcgaattt     2341 taacaaaata ttaacgctta caatttcctg atgcggtatt ttctccttac gcatctgtgc     2401 ggtatttcac accgcatacg cggatctgcg cagcaccatg gcctgaaata acctctgaaa     2461 gaggaacttg gttaggtacc ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt     2521 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca     2581 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa     2641 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc     2701 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg     2761 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg     2821 gaggcctagg cttttgcaaa aagcttaatt aactgttgac aattaatcat cggcatagta     2881 tatcggcata gtataatacg acaaggtgag gaactaaacc caggaggcag atcatgattg     2941 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg     3001 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg     3061 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg     3121 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg     3181 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc     3241 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc     3301 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc     3361 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc     3421 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg     3481 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct     3541 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt     3601 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc     3661 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt     3721 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc     3781 acgatggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg     3841 aatcgatagc gataaggatc ctctttgcgc ttgcgttttc ccttgtccag atagcccagt     3901 agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac ccggtatcag     3961 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca     4021 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt     4081 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc     4141 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct     4201 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg     4261 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca     4321 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact     4381 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta     4441 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta     4501 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct     4561 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt     4621 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atttcaagaa gatcctttga     4681 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca     4741 tgagattatc aaaaaggatc ttcacctaga tccttttata gtccggaaat acaggaacgc     4801 acgctggatg gcccttcgct gggatggtga aaccatgaaa aatggcagct tcagtggatt     4861 aagtgggggt aatgtggcct gtaccctctg gttgcatagg tattcatacg gttaaaattt     4921 atcaggcgcg attgcggcag tttttcgggt ggtttgttgc catttttacc tgtctgctgc     4981 cgtgatcgcg ctgaacgcgt tttagcggtg cgtacaatta agggattatg gtaaatccac     5041 ttactgtctg ccctcgtagc catcgagata aaccgcagta ctccggccac gatgcgtccg     5101 gcgtagagga tcgagatct