| 质粒名称: | pTriEx-mCherry-PA-Rac1-T17N |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 22029 |
| 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 |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 6000 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达，细菌表达，昆虫表达 |
| 筛选标记: | -- |
| 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: | PA-Rac1-T17N,LOV(Leu404-Leu546)-Rac1(Ile4)-T17N |
| 物种来源: | H. sapiens (human); Avena sativa (oat) |
| 插入大小: | 1100 bp |
| 基因突变: | Rac1起始于I4，包含突变T17N。 |
| GenBank编号 | AAA36544 |
| 启动子 | -- |
| 标签/融合蛋白: |
6X His（主干上的N终端）
mCherry（主干上的N终端）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | Bam HI(未破坏) |
| 3' 克隆位点: | HindIII (未破坏) |
| 5' 测序引物 | pTriExUP |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pTriEx-mCherry-PA-Rac1-T17N was a gift from Klaus Hahn (Addgene plasmid # 22029 ; http://n2t.net/addgene:22029 ; RRID:Addgene_22029) |
| 参考文献部分: | A genetically encoded photoactivatable Rac controls the motility of living cells. Wu YI, Frey D, Lungu OI, Jaehrig A, Schlichting I, Kuhlman B, Hahn KM. Nature. 2009 Sep 3. 461(7260):104-8. 10.1038/nature08241 PubMed 19693014 |
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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-mCherry-PA-Rac1-T17N 6745 bp ds-DNA circular SYN 26-MAR-2019
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pTriEx-mCherry-PA-Rac1-T17N
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6745)
AUTHORS Wu YI, Frey D, Lungu OI, Jaehrig A, Schlichting I, Kuhlman B, Hahn
KM
TITLE A genetically encoded photoactivatable Rac controls the motility of
living cells.
JOURNAL Nature. 2009 Sep 3. 461(7260):104-8.
PUBMED 19693014
REFERENCE 2 (bases 1 to 6745)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Mar 26, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6745
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 25..328
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 330..529
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 483..503
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
primer_bind 735..757
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 819..838
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
primer_bind 880..899
/label=T7
/note="T7 promoter, forward primer"
promoter 880..898
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 899..923
/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 939..1048
/label=p10 promoter
/note="baculovirus promoter for expression in insect cells"
CDS 1069..1086
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 1093..1797
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
GRHSTGGMDELYK"
primer_bind complement(1235..1253)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 1503..1522
/label=mCherry-F
/note="mCherry, forward primer"
CDS 2837..2869
/codon_start=1
/product="HSV (herpes simplex virus) epitope tag"
/label=HSV tag
/translation="QPELAPEDPED"
CDS 2876..2899
/codon_start=1
/product="8xHis affinity tag"
/label=8xHis
/translation="HHHHHHHH"
primer_bind complement(2989..3008)
/label=Bglob-pA-R
/note="Rabbit beta-globin polyA region, reverse primer"
polyA_site 3054..3109
/label=-beta-globin poly(A) signal
/note="rabbit beta-globin polyadenylation signal"
primer_bind complement(3108..3127)
/label=rbglobpA-R
/note="Rabbit beta-globin polyA, reverse primer. Also
called rb-glob-pA-term-R"
terminator 3197..3244
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
misc_recomb 3257..3962
/label=baculovirus recombination region (ORF1629)
/note="contains part of ORF1629"
3'UTR 3345..3717
/label=polyhedrin 3' UTR
/note="3' UTR from the Autographa californica
nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
CDS complement(3374..3962)
/codon_start=2
/product="baculovirus capsid-associated protein"
/label=ORF1629
/note="required for viral replication"
/translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
protein_bind 3971..4004
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
rep_origin complement(4172..4759)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4251..4270)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4772..5632)
/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 5395..5414
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
misc_recomb 5875..6702
/label=baculovirus recombination region (lef2/ORF603)
/note="contains ORF603 and part of lef2"
CDS 5875..5999
/codon_start=3
/product="baculovirus late expression factor 2"
/label=lef2
/translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
CDS complement(6037..6642)
/codon_start=1
/product="baculovirus ORF603 protein"
/label=ORF603
/translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
ORIGIN
1 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
61 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
121 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
181 agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg acggtaaatg
241 gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt ggcagtacat
301 ctacgtatta gtcatcgcta ttaccatgct gatgcggttt tggcagtaca ccaatgggcg
361 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
421 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc ccgccccgtt
481 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagac gtcgtttagt
541 gaaccgtcag atcactagat gctttattgc ggtagtttat cacagttaaa ttgctaacgc
601 cagtctcgaa cttaacgtgc agaagttggt cgtgaggcac tgggcaggta agtatcgggc
661 cctttgtgcg gggggagcgg ctcggggctg tccgcggggg gacggctgcc ttcggggggg
721 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
781 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct
841 gtctcatcat tttggcaaag aattggatcg gaccgaaatt aatacgactc actatagggg
901 aattgtgagc ggataacaat tccccggagt taatccggga cctttaattc aacccaacac
961 aatatattat agttaaataa gaattattat caaatcattt gtatattaat taaaatacta
1021 tactgtaaat tacattttat ttacaatcaa aggagatata ccatggcaca ccatcaccac
1081 catcacggat ctgtgagcaa gggcgaggag gataacatgg ccatcatcaa ggagttcatg
1141 cgcttcaagg tgcacatgga gggctccgtg aacggccacg agttcgagat cgagggcgag
1201 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
1261 cccctgccct tcgcctggga catcctgtcc cctcagttca tgtacggctc caaggcctac
1321 gtgaagcacc ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag
1381 tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc
1441 ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac
1501 ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct cctccgagcg gatgtacccc
1561 gaggacggcg ccctgaaggg cgagatcaag cagaggctga agctgaagga cggcggccac
1621 tacgacgctg aggtcaagac cacctacaag gccaagaagc ccgtgcagct gcccggcgcc
1681 tacaacgtca acatcaagtt ggacatcacc tcccacaacg aggactacac catcgtggaa
1741 cagtacgaac gcgccgaggg ccgccactcc accggcggca tggacgagct gtacaaggga
1801 tccttggcta ctacacttga acgtattgag aagaactttg tcattactga cccaagattg
1861 ccagataatc ccattatatt cgcgtccgat agtttcttgc agttgacaga atatagccgt
1921 gaagaaattt tgggaagaaa ctgcaggttt ctacaaggtc ctgaaactga tcgcgcgaca
1981 gtgagaaaaa ttagagatgc catagataac caaacagagg tcactgttca gctgattaat
2041 tatacaaaga gtggtaaaaa gttctggaac ctctttcact tgcagcctat gcgagatcag
2101 aagggagatg tccagtactt tattggggtt cagttggatg gaactgagca tgtccgagat
2161 gctgccgaga gagagggagt catgctgatt aagaaaactg cagaaaatat tgatgaggcg
2221 gcaaaagaac ttatcaagtg tgtggtggtg ggagacggag ctgtaggtaa aaattgccta
2281 ctgatcagtt acacaaccaa tgcatttcct ggagaatata tccctactgt ctttgacaat
2341 tattctgcca atgttatggt agatggaaaa ccggtgaatc tgggcttatg ggatacagct
2401 ggacaagaag attatgacag attacgcccc ctatcctatc cgcaaacaga tgtgttctta
2461 atttgctttt cccttgtgag tcctgcatca tttgaaaatg tccgtgcaaa gtggtatcct
2521 gaggtgcggc accactgtcc caacactccc atcatcctag tgggaactaa acttgatctt
2581 agggatgata aagacacgat cgagaaactg aaggagaaga agctgactcc catcacctat
2641 ccgcagggtc tagcaatggc taaggagatt ggtgctgtaa aatacctgga gtgctcggcg
2701 ctcacacagc gaggcctcaa gacagtgttt gacgaagcga tccgagcagt cctctgcccg
2761 cctcccgtga agaagaggaa gagaaaatgc ctgctgttgt aagcttgcgg ccgcacagct
2821 gtatacacgt gcaagccagc cagaactcgc cccggaagac cccgaggatc tcgagcacca
2881 ccatcaccat caccatcact aagtgattaa cctcaggtgc aggctgccta tcagaaggtg
2941 gtggctggtg tggccaatgc cctggctcac aaataccact gagatcgatc tttttccctc
3001 tgccaaaaat tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag
3061 gaaatttatt ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac
3121 atatgggagg gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac
3181 atatgcccat atgtaactag cataacccct tggggcctct aaacgggtct tgaggggttt
3241 tttgctgaaa gcatgcggag gaaattctcc ttgaagtttc cctggtgttc aaagtaaagg
3301 agtttgcacc agacgcacct ctgttcactg gtccggcgta ttaaaacacg atacattgtt
3361 attagtacat ttattaagcg ctagattctg tgcgttgttg atttacagac aattgttgta
3421 cgtattttaa taattcatta aatttataat ctttagggtg gtatgttaga gcgaaaatca
3481 aatgattttc agcgtcttta tatctgaatt taaatattaa atcctcaata gatttgtaaa
3541 ataggtttcg attagtttca aacaagggtt gtttttccga accgatggct ggactatcta
3601 atggattttc gctcaacgcc acaaaacttg ccaaatcttg tagcagcaat ctagctttgt
3661 cgatattcgt ttgtgttttg ttttgtaata aaggttcgac gtcgttcaaa atattatgcg
3721 cttttgtatt tctttcatca ctgtcgttag tgtacaattg actcgacgta aacacgttaa
3781 atagagcttg gacatattta acatcgggcg tgttagcttt attaggccga ttatcgtcgt
3841 cgtcccaacc ctcgtcgtta gaagttgctt ccgaagacga ttttgccata gccacacgac
3901 gcctattaat tgtgtcggct aacacgtccg cgatcaaatt tgtagttgag ctttttggaa
3961 ttgcgatcgc ataacttcgt atagcataca ttatacgaag ttataagctc ggaacgctgc
4021 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
4081 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
4141 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc ctgacgagca
4201 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
4261 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
4321 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
4381 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
4441 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
4501 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
4561 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
4621 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
4681 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
4741 cagaaaaaaa ggatctcaag aagatccttt gttaccaatg cttaatcagt gaggcaccta
4801 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
4861 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
4921 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
4981 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
5041 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
5101 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
5161 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
5221 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
5281 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
5341 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
5401 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
5461 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
5521 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
5581 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
5641 tttttcaata ttattgaagc atttatcagg gttattgtct catgtccgcg cgtttcctgc
5701 atcttttaat caaatcccaa gatgtgtata aacgcgccgg tatgtacagg aagaggttta
5761 tactaaactg ttacattgca aacgtggttt cgtgtgccaa gtgtgaaaac cgatgtttaa
5821 tcaaggctct gacgcatttc tacaaccacg actccaagtg tgtgggtgaa gtcatgcatc
5881 ttttaatcaa atcccaagat gtgtataaac caccaaactg ccaaaaaatg aaaactgtcg
5941 acaagctctg tccgtttgct ggcaactgca agggtctcaa tcctatttgt aattattgaa
6001 taataaaaca attataaatg tcaaatttgt tttttattaa cgatacaaac caaacgcaac
6061 aagaacattt gtagtattat ctataattga aaacgcgtag ttataatcgc tgaggtaata
6121 tttaaaatca ttttcaaatg attcacagtt aatttgcgac aatataattt tattttcaca
6181 taaactagac gccttgtcgt cttcttcttc gtattccttc tctttttcat ttttctcttc
6241 ataaaaatta acatagttat tatcgtatcc atatatgtat ctatcgtata gagtaaattt
6301 tttgttgtca taaatatata tgtctttttt aatggggtgt atagtaccgc tgcgcatagt
6361 ttttctgtaa tttacaacag tgctattttc tggtagttct tcggagtgtg ttgctttaat
6421 tattaaattt atataatcaa tgaatttggg atcgtcggtt ttgtacaata tgttgccggc
6481 atagtacgca gcttcttcta gttcaattac accatttttt agcagcaccg gattaacata
6541 actttccaaa atgttgtacg aaccgttaaa caaaaacagt tcacctccct tttctatact
6601 attgtctgcg agcagttgtt tgttgttaaa aataacagcc attgtaatga gacgcacaaa
6661 ctaatatcac aaactggaaa tgtctatcaa tatatagttg ctgatggccg gcctattaat
6721 agtaatcaat tacggggtca ttagt
//
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