| 质粒名称: | pNLF1W |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 136404 |
| 存储实验室: | Sebastian Maurer |
|
载体骨架基本信息 |
|
| 载体名称: | pNLF1-N CMV-hygro |
| 载体出品公司: | Promega |
| 载体抗性: | 氯霉素和氨苄青霉素 |
| 空载体大小: | 7662 bp |
| 完整质粒大小: | -- |
| 载体修饰: | attR1和attR2位点用于网关重组，ccdB盒和CmR用于细菌选择 |
| 载体类型: | 哺乳动物表达，荧光素酶 |
| 筛选标记: | 潮霉素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | -- |
| 物种来源: | -- |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | CMV |
| 标签/融合蛋白: | NanoLuc luciferase（插入物上的N末端） |
| 克隆方法: | Gateway克隆 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | TAATACGACTCACTATAGGG |
| 3' 测序引物 | CTAGTTATTGCTCAGCGG |
|
文献引用方法 |
|
| 材料和方法部分: | pNLF1W was a gift from Sebastian Maurer (Addgene plasmid # 136404 ; http://n2t.net/addgene:136404 ; RRID:Addgene_136404) |
| 参考文献部分: | rec-YnH enables simultaneous many-by-many detection of direct protein-protein and protein-RNA interactions. Yang JS, Garriga-Canut M, Link N, Carolis C, Broadbent K, Beltran-Sastre V, Serrano L, Maurer SP. Nat Commun. 2018 Sep 14;9(1):3747. doi: 10.1038/s41467-018-06128-x. 10.1038/s41467-018-06128-x PubMed 30217970 |
买家导航
pNLF1W载体质粒基本信息
pNLF1W质粒图谱载体图谱和pNLF1W载体序列质粒序列多克隆位点信息
pNLF1W质粒载体简介
pNLF1W质粒序列载体序列
LOCUS pNLF1W 7668 bp ds-DNA circular SYN 07-FEB-2020
DEFINITION Expression of N-terminal NanoLuc luciferase fused proteins in
mammalian cells. Genes can be cloned using Gateway cloning system.
Cloned vectors can be used for NanoBRET assay..
ACCESSION .
VERSION .
KEYWORDS pNLF1W
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7668)
AUTHORS Yang JS, Garriga-Canut M, Link N, Carolis C, Broadbent K,
Beltran-Sastre V, Serrano L, Maurer SP
TITLE rec-YnH enables simultaneous many-by-many detection of direct
protein-protein and protein-RNA interactions.
JOURNAL Nat Commun. 2018 Sep 14;9(1):3747. doi: 10.1038/s41467-018-06128-x.
PUBMED 30217970
REFERENCE 2 (bases 1 to 7668)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Feb 7, 2020 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7668
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 114..471
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(137..157)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 322..457
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 384..403
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 502..1539
/codon_start=1
/product="hygromycin B phosphotransferase"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
EVGRV"
RBS 1518..1526
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
polyA_signal 1563..1611
/note="synthetic polyadenylation signal"
promoter 1767..1871
/gene="bla"
/label=AmpR promoter
CDS 1872..2732
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(2090..2109)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin complement(2890..3478)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(2970..2989)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
misc_feature 3594..3877
/label=cer region
/note="ColE1-derived recombination site that helps to
maintain plasmids as monomers"
enhancer 4068..4447
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 4448..4651
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 4602..4622
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
intron 4787..4919
/label=chimeric intron
/note="chimera between introns from human beta-globin and
immunoglobulin heavy chain genes"
primer_bind 4902..4921
/label=pMT2-F
/note="Synthetic intron, forward primer"
primer_bind 4963..4982
/label=T7
/note="T7 promoter, forward primer"
promoter 4963..4981
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
CDS 4995..5507
/codon_start=1
/product="NanoLuc(R) luciferase"
/label=Nluc
/note="human codon-optimized"
/translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
RLCERILA"
protein_bind 5604..5728
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 5753..5783
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 5837..6496
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
primer_bind complement(5903..5922)
/label=CAT-R
/note="Chloramphenicol resistance gene, reverse primer"
CDS 6838..7143
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
primer_bind 7022..7041
/label=ccdB-fwd
/note="ccdB gene, forward primer"
protein_bind complement(7184..7308)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
primer_bind complement(7366..7384)
/label=T7 Term
/note="T7 terminator, reverse primer"
polyA_signal 7416..7537
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(7427..7446)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 7481..7500
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
ORIGIN
1 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gcggatctgc
61 gcagcaccat ggcctgaaat aacctctgaa agaggaactt ggttaggtac cttctgaggc
121 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
181 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
241 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
361 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcgat
481 tcttctgaca ctagcgccac catgaagaag cccgaactca ccgctaccag cgttgaaaaa
541 tttctcatcg agaagttcga cagtgtgagc gacctgatgc agttgtcgga gggcgaagag
601 agccgagcct tcagcttcga tgtcggcgga cgcggctatg tactgcgggt gaatagctgc
661 gctgatggct tctacaaaga ccgctacgtg taccgccact tcgccagcgc tgcactaccc
721 atccccgaag tgttggacat cggcgagttc agcgagagcc tgacatactg catcagtaga
781 cgcgcccaag gcgttactct ccaagacctc cccgaaacag agctgcctgc tgtgttacag
841 cctgtcgccg aagctatgga tgctattgcc gccgccgacc tcagtcaaac cagcggcttc
901 ggcccattcg ggccccaagg catcggccag tacacaacct ggcgggattt catttgcgcc
961 attgctgatc cccatgtcta ccactggcag accgtgatgg acgacaccgt gtccgccagc
1021 gtagctcaag ccctggacga actgatgctg tgggccgaag actgtcccga ggtgcgccac
1081 ctcgtccatg ccgacttcgg cagcaacaac gtcctgaccg acaacggccg catcaccgcc
1141 gtaatcgact ggtccgaagc tatgttcggg gacagtcagt acgaggtggc caacatcttc
1201 ttctggcggc cctggctggc ttgcatggag cagcagactc gctacttcga gcgccggcat
1261 cccgagctgg ccggcagccc tcgtctgcga gcctacatgc tgcgcatcgg cctggatcag
1321 ctctaccaga gcctcgtgga cggcaacttc gacgatgctg cctgggctca aggccgctgc
1381 gatgccatcg tccgcagcgg ggccggcacc gtcggtcgca cacaaatcgc tcgccggagc
1441 gcagccgtat ggaccgacgg ctgcgtcgag gtgctggccg acagcggcaa ccgccggccc
1501 agtacacgac cgcgcgctaa ggaggtaggt cgagtttaga ctctagccat cacgatggcc
1561 gcaataaaat atctttattt tcattacatc tgtgtgttgg ttttttgtgt gaatcgatag
1621 cgataaggat cctctttgcg cttgcgtttt cccttgtcca gatagcccag tagctgacat
1681 tcatccgggg tcagcaccgt ttctgcggac tggctttcta cgtaatggtt tcttagacgt
1741 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
1801 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
1861 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
1921 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
1981 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
2041 gttttcgccc cgaagaacgt tttccaatga tgagcacttt caaagttctg ctatgtggcg
2101 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
2161 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
2221 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
2281 tgacaactat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
2341 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
2401 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
2461 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
2521 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
2581 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
2641 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
2701 agataggtgc ctcactgatt aagcattggt aattcgaaat gaccgaccaa gcgacgccca
2761 accggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
2821 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
2881 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
2941 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
3001 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
3061 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
3121 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
3181 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
3241 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
3301 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa
3361 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaaaaaa ccaccgctgg
3421 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatttcaaga
3481 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
3541 gattttggtc atgagattat caaaaaggat cttcacctag atccttttat agtccggaaa
3601 tacaggaacg cacgctggat ggcccttcgc tgggatggtg aaaccatgaa aaatggcagc
3661 ttcagtggat taagtggggg taatgtggcc tgtaccctct ggttgcatag gtattcatac
3721 ggttaaaatt tatcaggcgc gattgcggca gtttttcggg tggtttgttg ccatttttac
3781 ctgtctgctg ccgtgatcgc gctgaacgcg ttttagcggt gcgtacaatt aagggattat
3841 ggtaaatcca cttactgtct gccctcgtag ccatcgagat aaaccgcagt actccggcca
3901 cgatgcgtcc ggcgtagagg atcgagatct tcaatattgg ccattagcca tattattcat
3961 tggttatata gcataaatca atattggcta ttggccattg catacgttgt atctatatca
4021 taatatgtac atttatattg gctcatgtcc aatatgaccg ccatgttggc attgattatt
4081 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
4141 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
4201 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
4261 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
4321 gccaagtccg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
4381 gtacatgacc ttacgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
4441 taccatggtg atgcggtttt ggcagtacac caatgggcgt ggatagcggt ttgactcacg
4501 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
4561 acgggacttt ccaaaatgtc gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg
4621 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tcactagaag
4681 ctttattgcg gtagtttatc acagttaaat tgctaacgca gtcagtgctt ctgacacaac
4741 agtctcgaac ttaagctgca gaagttggtc gtgaggcact gggcaggtaa gtatcaaggt
4801 tacaagacag gtttaaggag accaatagaa actgggcttg tcgagacaga gaagactctt
4861 gcgtttctga taggcaccta ttggtcttac tgacatccac tttgcctttc tctccacagg
4921 tgtccactcc cagttcaatt acagctctta aggctagagt attaatacga ctcactatag
4981 ggctagcgct caccatggtc ttcacactcg aagatttcgt tggggactgg cgacagacag
5041 ccggctacaa cctggaccaa gtccttgaac agggaggtgt gtccagtttg tttcagaatc
5101 tcggggtgtc cgtaactccg atccaaagga ttgtcctgag cggtgaaaat gggctgaaga
5161 tcgacatcca tgtcatcatc ccgtatgaag gtctgagcgg cgaccaaatg ggccagatcg
5221 aaaaaatttt taaggtggtg taccctgtgg atgatcatca ctttaaggtg atcctgcact
5281 atggcacact ggtaatcgac ggggttacgc cgaacatgat cgactatttc ggacggccgt
5341 atgaaggcat cgccgtgttc gacggcaaaa agatcactgt aacagggacc ctgtggaacg
5401 gcaacaaaat tatcgacgag cgcctgatca accccgacgg ctccctgctg ttccgagtaa
5461 ccatcaacgg agtgaccggc tggcggctgt gcgaacgcat tctggcgggc tcgagcggcg
5521 cgatcgcttc cgaattcaga gctcaaccgc ggatatctag atttgggccc aattcctgca
5581 ggattttgcg gccgcggatc tccacaagtt tgtacaaaaa agctgaacga gaaacgtaaa
5641 atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact
5701 gtaaaacaca acatatccag tcactatggc ggccgcatta ggcaccccag gctttacact
5761 ttatgcttcc ggctcgtata atgtgtggat tttgagttag gatccgtcga gattttcagg
5821 agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca
5881 atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca
5941 gaccgttcag ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt
6001 ttatccggcc tttattcaca ttcttgcccg cctgatgaat gctcatccgg aattccgtat
6061 ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt
6121 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca
6181 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc
6241 taaagggttt attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag
6301 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa
6361 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
6421 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg
6481 gcagggcggg gcgtaaacgc gtggatccgg cttactaaaa gccagataac agtatgcgta
6541 tttgcgcgct gatttttgcg gtataagaat atatactgat atgtataccc gaagtatgtc
6601 aaaaagaggt atgctatgaa gcagcgtatt acagtgacag ttgacagcga cagctatcag
6661 ttgctcaagg catatatgat gtcaatatct ccggtctggt aagcacaacc atgcagaatg
6721 aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg atggctgagg
6781 tcgcccggtt tattgaaatg aacggctctt ttgctgacga gaacaggggc tggtgaaatg
6841 cagtttaagg tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag
6901 agtgatatta ttgacacgcc cgggcgacgg atggtgatcc ccctggccag tgcacgtctg
6961 ctgtcagata aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg
7021 cgcatgatga ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct
7081 gatctcagcc accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata
7141 taaatgtcag gctcccttat acacagccag tctgcaggtc gaccatagtg actggatatg
7201 ttgtgtttta cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat
7261 atttatatca ttttacgttt ctcgttcagc tttcttgtac aaagtggtaa gtaactaaga
7321 tccgtcgacc tgcagcggcc gcgcttgctg agttggctgc tgccaccgct gagcaataac
7381 tagcataacc ccttggccgc ttcgagcaga catgataaga tacattgatg agtttggaca
7441 aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc
7501 tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt
7561 tatgtttcag gttcaggggg agatgtggga ggttttttta agcaagtaaa acctctacaa
7621 atgtggtaaa atcgaatttt aacaaaatat taacgcttac aatttcct
//
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pNLF1W质粒载体应用举例
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