| 质粒名称: | pRS426GFP-2×PH(PLCδ) |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 36092 |
| 存储实验室: | Scott Emr |
|
载体骨架基本信息 |
|
| 载体名称: | pRS426 |
| 载体出品公司: | -- |
| 空载体大小: | -- |
| 完整质粒大小: | -- |
| 载体修饰: | 为了构建酵母GFP表达载体（pGOGFP），将PRC1启动子和GFP编码序列亚克隆到PRS426的BamHI位点。为了创建PtdIns（4,5）P2特异性耀斑，我们将PLCδ1 PH域的两个串联拷贝（Kavran等人，1998年）融合到GFP中。PCR用于产生编码两个PLCδ1 PH结构域的序列，两侧为BamHI/EcoRI-orEcoRI/SalI限制性位点。这些PCR产物用BamHI/EcoRI-orEcoRI/SalI进行消化，并在框架内克隆到pGO35（Burd和Emr，1998），pGO35已被BglII和SalI切割，以产生含有GFP-2×PH（PLCδ）融合的质粒pRS426GFP-2×PH（PLCδ）。 |
| 载体类型: | 酵母表达 |
| 筛选标记: | URA3 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | XL1 Blue |
| 拷贝数: | -- |
|
插入基因信息 |
|
| 插入基因名称: | 2xPH(PLCdelta) fusion |
| 物种来源: | R. norvegicus (rat) |
| 插入大小: | -- |
| 基因突变: | 包含PLCδ的aa11-140（包括PH域） |
| GenBank编号 | -- |
| 启动子: | PRC1 |
| 标签/融合蛋白: | GFP（插入件上的N端子） |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BglII（在克隆过程中被销毁） |
| 3’克隆位点: | SalI (未破坏) |
| 5’测序引物 | T3 |
| 3’测序引物 | T7 |
|
文献引用方法 |
|
| 材料和方法部分: | pRS426GFP-2×PH(PLCδ) was a gift from Scott Emr (Addgene plasmid # 36092 ; http://n2t.net/addgene:36092 ; RRID:Addgene_36092) |
| 参考文献部分: | The yeast synaptojanin-like proteins control the cellular distribution of phosphatidylinositol (4,5)-bisphosphate. Stefan CJ, Audhya A, Emr SD. Mol Biol Cell. 2002 Feb;13(2):542-57. 10.1091/mbc.01-10-0476 PubMed 11854411 |
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
载体图谱.png)
载体简介
载体序列
LOCUS pRS426GFP-2×PH(PLC-delta) 7741 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pRS426GFP-2×PH(PLC-delta)
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7741)
AUTHORS Stefan CJ, Audhya A, Emr SD
TITLE The yeast synaptojanin-like proteins control the cellular
distribution of phosphatidylinositol (4,5)-bisphosphate.
JOURNAL Mol Biol Cell. 2002 Feb;13(2):542-57.
PUBMED 11854411
REFERENCE 2 (bases 1 to 7741)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7741
/organism="synthetic DNA construct"
/mol_type="other DNA"
protein_bind 91..112
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter 127..157
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 165..181
/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 170..192
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 189..205
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 189..205
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 224..244
/label=T3
/note="T3 promoter, forward primer"
promoter 226..244
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
primer_bind 281..297
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 281..297
/label=pBluescriptSK
/note="For pBluescript vector"
primer_bind complement(823..851)
/label=GFP-R
/note="GFP, reverse primer. Does NOT anneal to EGFP"
primer_bind 1446..1466
/label=GFP-F
/note="GFP, forward primer. Does NOT anneal to EGFP"
primer_bind complement(2398..2417)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(2399..2417)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(2427..2444)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(2427..2443)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2436..2458)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 2584..3039
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2671..2690)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 2881..2902
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(3170..3973)
/codon_start=1
/gene="S. cerevisiae URA3"
/product="orotidine-5'-phosphate decarboxylase, required
for uracil biosynthesis"
/label=URA3
/note="yeast auxotrophic marker, counterselectable with
5-fluoroorotic acid (5-FOA)"
/translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
promoter complement(3974..4189)
/gene="S. cerevisiae URA3"
/label=URA3 promoter
primer_bind complement(4213..4232)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4332..4354
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4392..4410)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
rep_origin 4451..5793
/label=2u ori
/note="yeast 2u plasmid origin of replication"
protein_bind 4783..4830
/label=FRT
/bound_moiety="FLP recombinase from the Saccharomyces
cerevisiae 2u plasmid"
/note="FLP-mediated recombination occurs in the 8-bp core
sequence TCTAGAAA (Turan and Bode, 2011)."
promoter 5820..5924
/gene="bla"
/label=AmpR promoter
CDS 5925..6785
/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(6143..6162)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6956..7544
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7445..7464
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 7698..7715
/label=L4440
/note="L4440 vector, forward primer"
ORIGIN
1 accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
61 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
121 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
181 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
241 aagggaacaa aagctggagc tccaccgcgg tggcggccgc tctagaacta gtggatcctt
301 ctgcacaaga agccatattg acagagcagt atgtgaggac ctttcttcta cacaatgtaa
361 cagtcaatgg ttgtagtcaa taccacgacc tttacgtgca gttttagagc gaaacttcgg
421 ttttttgaga cttacctcgt gtttgtgtct ccctgggaat ggagccgcgc cacccttgtc
481 tattatgtat agtcttattt aatcatagcg atgttggtca tccagtacac ttcggtagca
541 accttcgttt gtgattgtct tggtaattgc ttccaacaac tttatccatc attgagacag
601 gggccatatc acccgcgggg tctcaaagaa ggggcccact aataaaagca cgagataaga
661 atgccagcaa aaaagctccg aaataattct tttcgtcttc cctcctagtc ttaacaagac
721 aagagagaga gagagaaaga atactcacta gagattgttt cttttctact caacttaaag
781 tatacatacg ctatgggtaa aggtgtagaa cttttcactg gagttgtccc aattcttgtt
841 gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat
901 gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact acctgttcca
961 tggccaacac ttgtcactac ttttacgtat ggtgttcaat gcttttcaag atacccagat
1021 catatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaaaga
1081 actatatttt tcaaagatga cgggaactac aagacacgtg ctgaagtcaa gtttgaaggt
1141 gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt
1201 cttggacaca aattggaata caactataac tcacacaatg tatacatcat ggcagacaaa
1261 caaaagaatg gaatcaaagt taacttcaaa attagacaca acattgaaga tggaagcgtt
1321 caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca
1381 gacaaccatt acctgtccac acaatctgcc ctttcgaaag atcccaacga aaagagagac
1441 cacatggtcc ttcttgagtt tgtaacagct gctgggatta cacatggcat ggatgaacta
1501 tacaagtccg gactcagatc ccacgggctc caggatgacc cggaccttca ggcccttctg
1561 aagggcagcc agcttctgaa ggtgaagtcc agctcgtggc gtagggaacg cttctacaag
1621 ctacaggagg actgcaagac catctggcag gaatctcgaa aggtcatgag gtccccggag
1681 tcgcagctgt tctccatcga ggacattcag gaggtacgga tgggacaccg cacagaaggc
1741 ctggagaagt ttgcccgaga catccccgag gatcgatgct tctccattgt cttcaaggac
1801 cagcgcaaca ccctagacct cattgcccca tcaccagctg acgctcagca ctgggtgcag
1861 ggcctgcgca agatcatcca ccactccggc tccatggacc agcggcagaa ggcagcggcc
1921 gcaggattcc gcaagggcga attcgccctt gcggaatccg ccgcggcagc tcacgggctc
1981 caggatgacc cggaccttca ggcccttctg aagggcagcc agcttctgaa ggtgaagtcc
2041 agctcgtggc gtagggaacg cttctacaag ctacaggagg actgcaagac catctggcag
2101 gaatctcgaa aggtcatgag gtccccggag tcgcagctgt tctccatcga ggacattcag
2161 gaggtacgga tgggacaccg cacagaaggc ctggagaagt ttgcccgaga catccccgag
2221 gatcgatgct tctccattgt cttcaaggac cagcgcaaca ccctagacct cattgcccca
2281 tcaccagctg acgctcagca ctgggtgcag ggcctgcgca agatcatcca ccactccggc
2341 tccatggacc agcggcagaa gtagtcgacc tcgagggggg gcccggtacc caattcgccc
2401 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa
2461 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
2521 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
2581 tggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
2641 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
2701 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
2761 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
2821 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
2881 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
2941 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
3001 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc ctgatgcggt attttctcct
3061 tacgcatctg tgcggtattt cacaccgcat agggtaataa ctgatataat taaattgaag
3121 ctctaatttg tgagtttagt atacatgcat ttacttataa tacagttttt tagttttgct
3181 ggccgcatct tctcaaatat gcttcccagc ctgcttttct gtaacgttca ccctctacct
3241 tagcatccct tccctttgca aatagtcctc ttccaacaat aataatgtca gatcctgtag
3301 agaccacatc atccacggtt ctatactgtt gacccaatgc gtctcccttg tcatctaaac
3361 ccacaccggg tgtcataatc aaccaatcgt aaccttcatc tcttccaccc atgtctcttt
3421 gagcaataaa gccgataaca aaatctttgt cgctcttcgc aatgtcaaca gtacccttag
3481 tatattctcc agtagatagg gagcccttgc atgacaattc tgctaacatc aaaaggcctc
3541 taggttcctt tgttacttct tctgccgcct gcttcaaacc gctaacaata cctgggccca
3601 ccacaccgtg tgcattcgta atgtctgccc attctgctat tctgtataca cccgcagagt
3661 actgcaattt gactgtatta ccaatgtcag caaattttct gtcttcgaag agtaaaaaat
3721 tgtacttggc ggataatgcc tttagcggct taactgtgcc ctccatggaa aaatcagtca
3781 agatatccac atgtgttttt agtaaacaaa ttttgggacc taatgcttca actaactcca
3841 gtaattcctt ggtggtacga acatccaatg aagcacacaa gtttgtttgc ttttcgtgca
3901 tgatattaaa tagcttggca gcaacaggac taggatgagt agcagcacgt tccttatatg
3961 tagctttcga catgatttat cttcgtttcc tgcaggtttt tgttctgtgc agttgggtta
4021 agaatactgg gcaatttcat gtttcttcaa cactacatat gcgtatatat accaatctaa
4081 gtctgtgctc cttccttcgt tcttccttct gttcggagat taccgaatca aaaaaatttc
4141 aaggaaaccg aaatcaaaaa aaagaataaa aaaaaaatga tgaattgaaa aggtggtatg
4201 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
4261 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
4321 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
4381 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
4441 ttcttagtat gatccaatat caaaggaaat gatagcattg aaggatgaga ctaatccaat
4501 tgaggagtgg cagcatatag aacagctaaa gggtagtgct gaaggaagca tacgataccc
4561 cgcatggaat gggataatat cacaggaggt actagactac ctttcatcct acataaatag
4621 acgcatataa gtacgcattt aagcataaac acgcactatg ccgttcttct catgtatata
4681 tatatacagg caacacgcag atataggtgc gacgtgaaca gtgagctgta tgtgcgcagc
4741 tcgcgttgca ttttcggaag cgctcgtttt cggaaacgct ttgaagttcc tattccgaag
4801 ttcctattct ctagaaagta taggaacttc agagcgcttt tgaaaaccaa aagcgctctg
4861 aagacgcact ttcaaaaaac caaaaacgca ccggactgta acgagctact aaaatattgc
4921 gaataccgct tccacaaaca ttgctcaaaa gtatctcttt gctatatatc tctgtgctat
4981 atccctatat aacctaccca tccacctttc gctccttgaa cttgcatcta aactcgacct
5041 ctacattttt tatgtttatc tctagtatta ctctttagac aaaaaaattg tagtaagaac
5101 tattcataga gtgaatcgaa aacaatacga aaatgtaaac atttcctata cgtagtatat
5161 agagacaaaa tagaagaaac cgttcataat tttctgacca atgaagaatc atcaacgcta
5221 tcactttctg ttcacaaagt atgcgcaatc cacatcggta tagaatataa tcggggatgc
5281 ctttatcttg aaaaaatgca cccgcagctt cgctagtaat cagtaaacgc gggaagtgga
5341 gtcaggcttt ttttatggaa gagaaaatag acaccaaagt agccttcttc taaccttaac
5401 ggacctacag tgcaaaaagt tatcaagaga ctgcattata gagcgcacaa aggagaaaaa
5461 aagtaatcta agatgctttg ttagaaaaat agcgctctcg ggatgcattt ttgtagaaca
5521 aaaaagaagt atagattctt tgttggtaaa atagcgctct cgcgttgcat ttctgttctg
5581 taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt
5641 ttacaaaaat gaagcacaga ttcttcgttg gtaaaatagc gctttcgcgt tgcatttctg
5701 ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt
5761 ttgttctaca aaatgaagca cagatgcttc gttcaggtgg cacttttcgg ggaaatgtgc
5821 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
5881 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
5941 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
6001 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
6061 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
6121 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
6181 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
6241 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
6301 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
6361 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
6421 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
6481 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
6541 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
6601 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
6661 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
6721 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
6781 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
6841 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
6901 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
6961 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
7021 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
7081 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
7141 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
7201 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
7261 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
7321 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
7381 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
7441 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
7501 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
7561 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
7621 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
7681 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
7741 a
//
