| 质粒名称: | pTriEx-RhoA FLARE.sc Biosensor WT |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 12150 |
| 存储实验室: | Klaus Hahn |
|
载体骨架基本信息 |
|
| 载体名称: | pTriEx |
| 载体出品公司: | Novagen |
| 空载体大小: | 5000 bp |
| 完整质粒大小: | 7323bp |
| 载体类型: | 哺乳细胞表达、细菌细胞表达、昆虫细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | RhoA Biosensor / 6xHis-RBD-CFP-Linker-Citrine-RhoA WT / RhoA |
| 物种来源: | H. sapiens (human) |
| 插入大小: | 2400 bp |
| 标签/融合蛋白: | 6xHis (N terminal on insert) |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | NcoI(未破坏) |
| 3’克隆位点: | XhoI (未破坏) |
|
文献引用方法 |
|
| 材料和方法部分: | pTriEx-RhoA FLARE.sc Biosensor WT was a gift from Klaus Hahn (Addgene plasmid # 12150 ; http://n2t.net/addgene:12150 ; RRID:Addgene_12150) |
| 参考文献部分: | Spatiotemporal dynamics of RhoA activity in migrating cells. Pertz O, Hodgson L, Klemke RL, Hahn KM. Nature. 2006 Apr 20. 440(7087):1069-72. 10.1038/nature04665 PubMed 16547516 |
买家导航
载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pTriEx-RhoA FLARE.sc Biosensor WT 7323 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pTriEx-RhoA FLARE.sc Biosensor WT
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7323)
AUTHORS Pertz O, Hodgson L, Klemke RL, Hahn KM
TITLE Spatiotemporal dynamics of RhoA activity in migrating cells.
JOURNAL Nature. 2006 Apr 20. 440(7087):1069-72.
PUBMED 16547516
REFERENCE 2 (bases 1 to 7323)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..7323
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 1..304
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 306..505
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 459..479
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 711..733
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 795..814
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
primer_bind 856..875
/label=T7
/note="T7 promoter, forward primer"
promoter 856..874
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 875..899
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter 915..1024
/label=p10 promoter
/note="baculovirus promoter for expression in insect cells"
CDS 1045..1062
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 1327..2043
/codon_start=1
/product="cyan variant of GFP"
/label=CFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 2116..2832
/codon_start=1
/product="enhanced variant of YFP (Heikal et al., 2001)"
/label=Citrine
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
CDS 2839..3420
/codon_start=1
/product="human Rho family small GTPase RhoA"
/label=RhoA
/translation="MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVAD
IEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVK
HFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKT
KDGVREVFEMATRAALQARRGKKKSGCLVL"
CDS 3430..3453
/codon_start=1
/product="8xHis affinity tag"
/label=8xHis
/translation="HHHHHHHH"
primer_bind complement(3543..3562)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_signal 3608..3663
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal (Gil and
Proudfoot, 1987)"
primer_bind complement(3662..3681)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
terminator 3751..3798
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
misc_recomb 3811..4516
/label=baculovirus recombination region (ORF1629)
/note="contains part of ORF1629"
3'UTR 3899..4271
/label=polyhedrin 3' UTR
/note="3' UTR from the Autographa californica
nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
CDS complement(3928..4516)
/codon_start=2
/product="baculovirus capsid-associated protein"
/label=ORF1629
/note="required for viral replication"
/translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
protein_bind 4525..4558
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
rep_origin complement(4726..5313)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4805..4824)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(5326..6186)
/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 5949..5968
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
misc_recomb 6429..7256
/label=baculovirus recombination region (lef2/ORF603)
/note="contains ORF603 and part of lef2"
CDS 6429..6553
/codon_start=3
/product="baculovirus late expression factor 2"
/label=lef2
/translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
CDS complement(6591..7196)
/codon_start=1
/product="baculovirus ORF603 protein"
/label=ORF603
/translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
ORIGIN
1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
181 aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
241 catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
301 catgctgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg
361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
421 ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt
481 acggtgggag gtctatataa gcagacgtcg tttagtgaac cgtcagatca ctagatgctt
541 tattgcggta gtttatcaca gttaaattgc taacgccagt ctcgaactta acgtgcagaa
601 gttggtcgtg aggcactggg caggtaagta tcgggccctt tgtgcggggg gagcggctcg
661 gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt
721 ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt
781 cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt
841 ggatcggacc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
901 cggagttaat ccgggacctt taattcaacc caacacaata tattatagtt aaataagaat
961 tattatcaaa tcatttgtat attaattaaa atactatact gtaaattaca ttttatttac
1021 aatcaaagga gatataccat ggcacaccat caccaccatc acggtagtgg catcctggag
1081 gacctcaata tgctctacat ccggcagatg gcactcagcc tggaggacac agagctgcag
1141 aggaaactag atcatgagat ccggatgagg gatggggcct gcaagctgct ggcagcctgc
1201 tcccagcgag agcaggctct ggaagccacc aagagcctgc tggtgtgcaa cagccgtatt
1261 ctcagctaca tgggtgagct gcagcggcga aaggaggccc aggtgctgga gaagacaggc
1321 gggggaatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
1381 ctggacggcg acgtaaacgg ccacaggttc agcgtgtccg gcgagggcga gggcgatgcc
1441 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
1501 cccaccctcg tgaccaccct gacctggggc gtgcagtgct tcagccgcta ccccgaccac
1561 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgtacc
1621 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
1681 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
1741 gggcacaagc tggagtacaa ctacatcagc cacaacgtct atatcaccgc cgacaagcag
1801 aagaacggca tcaaggccca cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
1861 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
1921 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
1981 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
2041 aagggatcta cttctggttc tggtaaacct ggttctggtg aaggttctac taaaggtggt
2101 agtggcgcgg ccgctatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
2161 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
2221 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
2281 gtgccctggc ccaccctcgt gaccaccttc ggctacggcc tgatgtgctt cgcccgctac
2341 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
2401 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
2461 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
2521 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
2581 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
2641 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
2701 ctgcccgaca accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag
2761 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
2821 gagctgtaca aggggggaat ggctgccatc cggaagaaac tggtgattgt tggtgatgga
2881 gcctgtggaa agacatgctt gctcatagtc ttcagcaagg accagttccc agaggtgtat
2941 gtgcccacag tgtttgagaa ctatgtggca gatatcgagg tggatggaaa gcaggtagag
3001 ttggctttgt gggacacagc tgggcaggaa gattatgatc gcctgaggcc cctctcctac
3061 ccagataccg atgttatact gatgtgtttt tccatcgaca gccctgatag tttagaaaac
3121 atcccagaaa agtggacccc agaagtcaag catttctgtc ccaacgtgcc catcatcctg
3181 gttgggaata agaaggatct tcggaatgat gagcacacaa ggcgggagct agccaagatg
3241 aagcaggagc cggtgaaacc tgaagaaggc agagatatgg caaacaggat tggcgctttt
3301 gggtacatgg agtgttcagc aaagaccaaa gatggagtga gagaggtttt tgaaatggct
3361 acgagagctg ctctgcaagc tagacgtggg aagaaaaaat ctggttgcct tgtcttgtga
3421 taactcgagc accaccatca ccatcaccat cactaagtga ttaacctcag gtgcaggctg
3481 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
3541 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
3601 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
3661 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
3721 tagagtttgg caacatatgc ccatatgtaa ctagcataac cccttggggc ctctaaacgg
3781 gtcttgaggg gttttttgct gaaagcatgc ggaggaaatt ctccttgaag tttccctggt
3841 gttcaaagta aaggagtttg caccagacgc acctctgttc actggtccgg cgtattaaaa
3901 cacgatacat tgttattagt acatttatta agcgctagat tctgtgcgtt gttgatttac
3961 agacaattgt tgtacgtatt ttaataattc attaaattta taatctttag ggtggtatgt
4021 tagagcgaaa atcaaatgat tttcagcgtc tttatatctg aatttaaata ttaaatcctc
4081 aatagatttg taaaataggt ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat
4141 ggctggacta tctaatggat tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag
4201 caatctagct ttgtcgatat tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt
4261 caaaatatta tgcgcttttg tatttctttc atcactgtcg ttagtgtaca attgactcga
4321 cgtaaacacg ttaaatagag cttggacata tttaacatcg ggcgtgttag ctttattagg
4381 ccgattatcg tcgtcgtccc aaccctcgtc gttagaagtt gcttccgaag acgattttgc
4441 catagccaca cgacgcctat taattgtgtc ggctaacacg tccgcgatca aatttgtagt
4501 tgagcttttt ggaattgcga tcgcataact tcgtatagca tacattatac gaagttataa
4561 gctcggaacg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
4621 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
4681 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
4741 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
4801 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
4861 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
4921 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
4981 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
5041 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
5101 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
5161 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
5221 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
5281 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgttacc aatgcttaat
5341 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
5401 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
5461 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
5521 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
5581 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
5641 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
5701 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
5761 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
5821 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
5881 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
5941 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
6001 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
6061 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
6121 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
6181 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgtc
6241 cgcgcgtttc ctgcatcttt taatcaaatc ccaagatgtg tataaacgcg ccggtatgta
6301 caggaagagg tttatactaa actgttacat tgcaaacgtg gtttcgtgtg ccaagtgtga
6361 aaaccgatgt ttaatcaagg ctctgacgca tttctacaac cacgactcca agtgtgtggg
6421 tgaagtcatg catcttttaa tcaaatccca agatgtgtat aaaccaccaa actgccaaaa
6481 aatgaaaact gtcgacaagc tctgtccgtt tgctggcaac tgcaagggtc tcaatcctat
6541 ttgtaattat tgaataataa aacaattata aatgtcaaat ttgtttttta ttaacgatac
6601 aaaccaaacg caacaagaac atttgtagta ttatctataa ttgaaaacgc gtagttataa
6661 tcgctgaggt aatatttaaa atcattttca aatgattcac agttaatttg cgacaatata
6721 attttatttt cacataaact agacgccttg tcgtcttctt cttcgtattc cttctctttt
6781 tcatttttct cttcataaaa attaacatag ttattatcgt atccatatat gtatctatcg
6841 tatagagtaa attttttgtt gtcataaata tatatgtctt ttttaatggg gtgtatagta
6901 ccgctgcgca tagtttttct gtaatttaca acagtgctat tttctggtag ttcttcggag
6961 tgtgttgctt taattattaa atttatataa tcaatgaatt tgggatcgtc ggttttgtac
7021 aatatgttgc cggcatagta cgcagcttct tctagttcaa ttacaccatt ttttagcagc
7081 accggattaa cataactttc caaaatgttg tacgaaccgt taaacaaaaa cagttcacct
7141 cccttttcta tactattgtc tgcgagcagt tgtttgttgt taaaaataac agccattgta
7201 atgagacgca caaactaata tcacaaactg gaaatgtcta tcaatatata gttgctgatg
7261 gccggcctat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
7321 ccg
//
