| 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 |
买家导航
pFC32A载体基本信息
pFC32A载体图谱质粒图谱和多克隆位点信息
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
//
