pFC32A Nluc-CMV-Hygro-Flexi

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pFC32A载体基本信息

出品公司: Promega
载体名称: pFC32A ; pFC32A Nluc-CMV-Hygro-Flexi
质粒类型: 哺乳动物载体;NanoLuc素酶报告载体
高拷贝/低拷贝: --
克隆方法: 限制性内切酶,多克隆位点
启动子: CMV IE
载体大小: 6299 bp
5' 测序引物及序列: T7 Forward:TAATACGACTCACTATAGGG
3' 测序引物及序列: --
载体标签: NanoLuc
载体抗性: 氨苄青霉素
筛选标记: 潮霉素(Hygromycin)
克隆菌株: TOP10等常规菌株
宿主细胞(系): 常规哺乳动物细胞等
备注: pFC32A Nluc-CMV-Hygro-Flexi载体表达NanoLuc融合蛋白;NanoLuc萤光素酶(Nluc)更小(19.1kDa)、更亮(比萤火虫和海肾萤光素酶亮度高 100 倍)。
产品目录号: N1331
稳定性: 瞬表达或稳表达
组成型/诱导型: 组成型
病毒/非病毒: 非病毒

pFC32A载体质粒图谱和多克隆位点信息

pFC32A载体图谱



pFC32A Nluc CMV-Hygro Flexi载体图谱



CMV immediate early enhancer/promoter                        1–742
Chimeric intron                                              857–989
T7 RNA polymerase promoter (–17 to +3)                       1033–1052
SgfI                                                         1056–1063
Barnase coding region                                        1087–1422
EcoICRI                                                      1442–1447
Linker                                                       1447–1461
NanoLuc® protein coding region                               1462–1971
SV40 late poly(A) signal                                     2108–2329
SV40 early enhancer/promoter                                 2428–2840
Synthetic hygromycin (Hygr) coding region                    2871–3908
Synthetic poly(A) signal                                     3932–3980
β-lactamase (Ampr) coding region                             4241–5101
ColE1-derived plasmid origin of replication                  5256–5292

pFC32A载体简介

NanoLuc萤光素酶融合表达载体pFC32A Nluc CMV-Hygro Flexi(R) Vector

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、高效和高保真。

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

pFC32A载体序列

LOCUS       KF811455                6299 bp    DNA     circular SYN 03-FEB-2014
DEFINITION  NLuc CMV-Hygro Flexi Vector pFC32A, complete sequence.
ACCESSION   KF811455
VERSION     KF811455.1  GI:576890560
KEYWORDS    .
SOURCE      NLuc CMV-Hygro Flexi Vector pFC32A
  ORGANISM  NLuc CMV-Hygro Flexi Vector pFC32A
            other sequences; artificial sequences; vectors.
REFERENCE   1  (bases 1 to 6299)
  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, USA
FEATURES             Location/Qualifiers
     source          1..6299
                     /organism="NLuc CMV-Hygro Flexi Vector pFC32A"
                     /mol_type="other DNA"
                     /db_xref="taxon:1454398"
     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="SgfI site"
     CDS             1087..1422
                     /codon_start=1
                     /transl_table=11
                     /product="barnase"
                     /protein_id="AHH41351.1"
                     /db_xref="GI:576890562"
                     /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="AHH41352.1"
                     /db_xref="GI:576890563"
                     /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"
     gene            2871..3908
                     /gene="hygR"
     CDS             2871..3908
                     /gene="hygR"
                     /codon_start=1
                     /transl_table=11
                     /product="hygromycin resistance protein"
                     /protein_id="AHH41350.1"
                     /db_xref="GI:576890561"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQD
                     LPETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVY
                     HWQTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWS
                     EAMFGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQ
                     SLVDGNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPS
                     TRPRAKEVGRV"
     regulatory      3932..3980
                     /regulatory_class="polyA_signal_sequence"
                     /note="synthetic polyA signal"
     gene            4241..5101
                     /gene="ampR"
     CDS             4241..5101
                     /gene="ampR"
                     /function="resistance to ampicillin"
                     /note="ampicillin-resistance protein"
                     /codon_start=1
                     /transl_table=11
                     /product="beta-lactamase"
                     /protein_id="AHH41353.1"
                     /db_xref="GI:576890564"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
     rep_origin      5256..5292
                     /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 aagctcgatt cttctgacac tagcgccacc atgaagaagc
     2881 ccgaactcac cgctaccagc gttgaaaaat ttctcatcga gaagttcgac agtgtgagcg
     2941 acctgatgca gttgtcggag ggcgaagaga gccgagcctt cagcttcgat gtcggcggac
     3001 gcggctatgt actgcgggtg aatagctgcg ctgatggctt ctacaaagac cgctacgtgt
     3061 accgccactt cgccagcgct gcactaccca tccccgaagt gttggacatc ggcgagttca
     3121 gcgagagcct gacatactgc atcagtagac gcgcccaagg cgttactctc caagacctcc
     3181 ccgaaacaga gctgcctgct gtgttacagc ctgtcgccga agctatggat gctattgccg
     3241 ccgccgacct cagtcaaacc agcggcttcg gcccattcgg gccccaaggc atcggccagt
     3301 acacaacctg gcgggatttc atttgcgcca ttgctgatcc ccatgtctac cactggcaga
     3361 ccgtgatgga cgacaccgtg tccgccagcg tagctcaagc cctggacgaa ctgatgctgt
     3421 gggccgaaga ctgtcccgag gtgcgccacc tcgtccatgc cgacttcggc agcaacaacg
     3481 tcctgaccga caacggccgc atcaccgccg taatcgactg gtccgaagct atgttcgggg
     3541 acagtcagta cgaggtggcc aacatcttct tctggcggcc ctggctggct tgcatggagc
     3601 agcagactcg ctacttcgag cgccggcatc ccgagctggc cggcagccct cgtctgcgag
     3661 cctacatgct gcgcatcggc ctggatcagc tctaccagag cctcgtggac ggcaacttcg
     3721 acgatgctgc ctgggctcaa ggccgctgcg atgccatcgt ccgcagcggg gccggcaccg
     3781 tcggtcgcac acaaatcgct cgccggagcg cagccgtatg gaccgacggc tgcgtcgagg
     3841 tgctggccga cagcggcaac cgccggccca gtacacgacc gcgcgctaag gaggtaggtc
     3901 gagtttagac tctagccatc acgatggccg caataaaata tctttatttt cattacatct
     3961 gtgtgttggt tttttgtgtg aatcgatagc gataaggatc ctctttgcgc ttgcgttttc
     4021 ccttgtccag atagcccagt agctgacatt catccggggt cagcaccgtt tctgcggact
     4081 ggctttctac gtaatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg
     4141 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     4201 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     4261 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     4321 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     4381 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     4441 gagcactttc aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     4501 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     4561 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     4621 gagtgataac actgcggcca acttacttct gacaactatc ggaggaccga aggagctaac
     4681 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     4741 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     4801 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     4861 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     4921 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     4981 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     5041 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     5101 attcgaaatg accgaccaag cgacgcccaa ccggtatcag ctcactcaaa ggcggtaata
     5161 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5221 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5281 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5341 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5401 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5461 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5521 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5581 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5641 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     5701 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5761 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5821 attacgcgca gaaaaaaagg atttcaagaa gatcctttga tcttttctac ggggtctgac
     5881 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5941 ttcacctaga tccttttata gtccggaaat acaggaacgc acgctggatg gcccttcgct
     6001 gggatggtga aaccatgaaa aatggcagct tcagtggatt aagtgggggt aatgtggcct
     6061 gtaccctctg gttgcatagg tattcatacg gttaaaattt atcaggcgcg attgcggcag
     6121 tttttcgggt ggtttgttgc catttttacc tgtctgctgc cgtgatcgcg ctgaacgcgt
     6181 tttagcggtg cgtacaatta agggattatg gtaaatccac ttactgtctg ccctcgtagc
     6241 catcgagata aaccgcagta ctccggccac gatgcgtccg gcgtagagga tcgagatct
//

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