| 质粒名称: | p1xNLS-FRB-mCherry-hsp86-BSD |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 85795 |
| 存储实验室: | Tobias Spielmann |
|
载体骨架基本信息 |
|
| 载体名称: | pGEM |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 恶性疟原虫表达 |
| 筛选标记: | 杀稻瘟菌素 |
| 载体抗性: | 氨苄霉素,100 μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | -- |
|
插入基因信息 |
|
| 插入基因名称: | 1xNLS Mislocaliser |
| 物种来源: | Synthetic |
| 插入大小: | -- |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | Pfhsp86 |
| 标签/融合蛋白: | FRB*, mCherry |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | XhoI (未破坏) |
| 3’克隆位点: | XmaI (未破坏) |
| 5’测序引物 | hsp86fw |
| 3’测序引物 | pARLrv |
|
文献引用方法 |
|
| 材料和方法部分: | p1xNLS-FRB-mCherry-hsp86-BSD was a gift from Tobias Spielmann (Addgene plasmid # 85795 ; http://n2t.net/addgene:85795 ; RRID:Addgene_85795) |
| 参考文献部分: | A genetic system to study Plasmodium falciparum protein function. Birnbaum J, Flemming S, Reichard N, Soares AB, Mesen-Ramirez P, Jonscher E, Bergmann B, Spielmann T. Nat Methods. 2017 Mar 13. doi: 10.1038/nmeth.4223. 10.1038/nmeth.4223 PubMed 28288121 |
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 p1xNLS-FRB-mCherry-hsp86-BSD 7485 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expression of 1xNLS mislocaliser in P. falciparum.
ACCESSION .
VERSION .
KEYWORDS p1xNLS-FRB-mCherry-hsp86-BSD
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7485)
AUTHORS Birnbaum J, Flemming S, Reichard N, Soares AB, Mesen-Ramirez P,
Jonscher E, Bergmann B, Spielmann T
TITLE A genetic system to study Plasmodium falciparum protein function.
JOURNAL Nat Methods. 2017 Mar 13. doi: 10.1038/nmeth.4223.
PUBMED 28288121
REFERENCE 2 (bases 1 to 7485)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7485
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS 1357..1377
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1462..1740
/codon_start=1
/product="T2098L mutant of FKBP-rapamycin binding domain of
human FRAP"
/label=FRB*
/note="binds synthetic ligands such as AP21967"
/translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
CDS 1801..2508
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
primer_bind complement(1946..1964)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 2214..2233
/label=mCherry-F
/note="mCherry, forward primer"
CDS 3827..4225
/codon_start=1
/gene="Aspergillus terreus BSD"
/product="blasticidin S deaminase"
/label=BSD
/note="confers resistance to blasticidin"
/translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
KAIVKDSDGQPTAVGIRELLPSGYVWEG"
promoter complement(4803..4820)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(4827..4844)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(4827..4843)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(4836..4858)
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind complement(5052..5071)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5171..5193
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5231..5249)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5317..5421
/gene="bla"
/label=AmpR promoter
CDS 5422..6282
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMPTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(5640..5659)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6453..7041
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6942..6961
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 7195..7212
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 7329..7350
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 7365..7395
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 7403..7419
/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)."
primer_bind 7408..7430
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 7427..7443
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 7427..7443
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
promoter 7461..7479
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 7461..7478
/label=SP6
/note="SP6 promoter, forward primer"
ORIGIN
1 cggccgctaa cgtaacagac ttaggaggag atctagatct ttcatatata taatatttta
61 aaggggtaaa aaaaaaaata tatataataa taacaaataa ataaataaag gaaaacaata
121 caatacaata taatataatt aattttatgt atatattaag aaatatctta aaaaaaaaaa
181 aaaaaaatct gtttcatata aagaatatat aatatttata aaacgatgtt attcttataa
241 aaaaaaaaaa agaaaaaaaa aaataaaaaa taaaaggata ccccacctag tcaaaaataa
301 ataatattat atgtattaaa ataatattat gggtaattta atattatata tatatatata
361 tatatatata atttaagttt ttgtagatta tatataatat ataatatatt atatattata
421 tatatatata taaatatata ttgaaggtgt tttaaaaaaa aaaaaaaagc cccgcggaaa
481 ggggccattg gatatatatt tagtattcca tagtatatag ttaaatataa taatattata
541 gtaaattttt gcatgaattt aaagaaaaaa aaaaaaaaaa taacaaaaag gagtattata
601 taaatataac aaaataataa atactatacc tatatatata tatatatata tatacatata
661 tataaatata taaatatata aatatattta tataaaaata atattttata tataatatta
721 tatttttata atatcttttt ttttttaaat atcatctttg tttctttctt tctttctttc
781 tttctttctt tttttttttt tttgatttat atattatttt attttgtatt tacaaaatat
841 aataaaataa tttcatgttt agcaaaaatt ttaaataaaa aataataata taaaataaaa
901 agcatcattt aatataaaat aataaaatta aaataaaata agataattac caaaaataat
961 aaaaaaaatt aaaaagtatt aaaaaatata tatcatatat aaatatataa attttaacaa
1021 cgtgagaaat taagaaaaaa attatattta tataaataat ataatattta atatatatct
1081 catataatat tatatatata tatatatata tatatatatt ttttaaaaaa aaagaaaaaa
1141 taagtttatt tttacctttt aataaaaaca tatttttaga tatacataaa aatttttata
1201 ttattttatt ggtatatata tatatatata tatatataat aatatatata taataaaata
1261 tataaatatt tacataaata ttttacttta aaaagaaatt ttcctttttt attatatttt
1321 ttttcttccc acatttcgaa taaactcgag atggcaccaa aaaaaaaaag aaaagttacg
1381 cgtgatccaa caagaagtgc aaatagtgga gcaggagcag gagcaggagc aatattaagt
1441 agagctagca tggcttctag aatcctctgg catgagatgt ggcatgaagg cctggaagag
1501 gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc
1561 ttgcatgcta tgatggaacg gggcccccag actctgaagg aaacatcctt taatcaggcc
1621 tatggtcgag atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat
1681 gtcaaggacc tcctccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag
1741 actagtacaa gttatccata tgataatcca gattatgcac cagttgcaac attaggtacc
1801 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag
1861 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
1921 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
1981 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac
2041 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc
2101 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac
2161 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
2221 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc
2281 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct
2341 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc
2401 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa
2461 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagcc cgggtcgagg
2521 gatatggcag cttaatgttc gtttttctta tttatatatt tataccaatt gattgtattt
2581 ataactgtaa aaatgtgtat gttgtgtgca tatttttttt tgtgcatgca catgcatgta
2641 aatagctaaa attatgaaca ttttattttt tgttcagaaa aaaaaaactt tacacacata
2701 aaatggctag tatgaatagc catattttat ataaattaaa tcctatgaat ttatgaccat
2761 attaaaaatt tagatattta tggaacataa tatgtttgaa acaataagac aaaattatta
2821 ttattattat tatttttact gttataatta tgtgtctcct tcaatgattc ataaatagtt
2881 ggacttgatt tttaaaatgt ttataatatg attagcatag ttaaataaaa aaagttgaaa
2941 aattaaaaaa aaacatataa acacaaatga tggtttttcc ttcaatttcg atatcaattt
3001 atagaaacaa aatatatact tgtataattt tattttttta tataaatcat tacatatata
3061 attatacaat attttttcta agagataatt atatattaat atatataaaa aaaggtgttt
3121 tttttttttt tttttatttt tatttttatt ttatggtaat attttatttt ccttatttta
3181 taaattatat tagtttatat gtgattaatt ttatatatta tcaatttata tatttttaaa
3241 tgcttactta attatctttt tttttttttt tttttttttt tcccctcttt ttatattaat
3301 ttatttttga aaaaattgat atatatatat atatataata tatatatata catgtagtag
3361 tattaaacaa tgtataatat atataaataa tatatttata tatttcattt caattttaat
3421 tttttttggt tttttttttt tttctttttg tcatatttaa aaaaaattat attcatataa
3481 gttatgcatt ttttataaac attattcaat atatgtataa tataatatat atatatatat
3541 taatgtatta ttccaatgtg catgataaaa gaaaaaaata atatttataa aaaaaaagaa
3601 aaataaaaca aaaaaagaaa aaaaaaaaaa aaaaaaaaaa aatacaaaaa taaataatat
3661 aatttataat tatatattct tgtcacaata aaaatatata tatatatata tatatttata
3721 atatgtatat tttaaactag aaaaggaata actaatattt tatttattat cattcaagat
3781 ttatatttta taataataaa tacctaatag aaatatatca ggatccatgg ccaagccttt
3841 gtctcaagaa gaatccaccc tcattgaaag agcaacggct acaatcaaca gcatccccat
3901 ctctgaagac tacagcgtcg ccagcgcagc tctctctagc gacggccgca tcttcactgg
3961 tgtcaatgta tatcatttta ctgggggacc ttgtgcagaa ctcgtggtgc tgggcactgc
4021 tgctgctgcg gcagctggca acctgacttg tatcgtcgcg atcggaaatg agaacagggg
4081 catcttgagc ccctgcggac ggtgccgaca ggtgcttctc gatctgcatc ctgggatcaa
4141 agcgatagtg aaggacagtg atggacagcc gacggcagtt gggattcgtg aattgctgcc
4201 ctctggttat gtgtgggagg gctaaaagct tatttaataa tagattaaaa atattataaa
4261 aataaaaaca taaacacaga aattacaaaa aaaatacata tgaatttttt tttgtaatct
4321 tccttataaa tatagaataa tgaatcatat aaaacatatc attattcatt tatttacatt
4381 taaaattatt gtttcagtat ctttaattta ttatgtatat ataaaaataa cttacaattt
4441 tattaataaa caatatatgt ttattaattc atgttttgta atttatggga tagcgatttt
4501 ttttactgtc tgtatttttc ttttttaatt atgttttaat tgtattttat ttttattatt
4561 gttcttttta tagtattatt ttaaaacaaa atgtattttc taagaactta taataataat
4621 aaatataaat tttaataaaa attatattta tcttttacaa tatgaacata aagtacaaca
4681 ttaatatata gcttttaata tttttattcc taatcatgta aatcttaaat ttttcttttt
4741 aaacatatgt taaatattta tttctcatta tatataagaa catatttatt aaatctagaa
4801 ttctatagtg agtcgtatta caattcactg gccgtcgttt tacaacgtcg tgactgggaa
4861 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
4921 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
4981 tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
5041 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
5101 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
5161 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
5221 agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt
5281 tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
5341 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
5401 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
5461 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
5521 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
5581 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
5641 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
5701 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
5761 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
5821 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
5881 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
5941 gacgagcgtg acaccacgat gcctgtagca atgccaacaa cgttgcgcaa actattaact
6001 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
6061 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
6121 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
6181 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
6241 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
6301 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
6361 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
6421 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
6481 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
6541 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
6601 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
6661 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
6721 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
6781 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
6841 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
6901 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
6961 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
7021 ggggggcgga gcctatcgaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
7081 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
7141 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
7201 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
7261 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
7321 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
7381 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
7441 gaccatgatt acgccaagct atttaggtga cactatagaa tactc
//
