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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20℃
- 保质期：
6个月
- 英文名：
pHIL-S1
- 库存：
大量
- 供应商：
钰博生物
- 规格：
1ug/5ug
pHIL-S1
载体基本信息
| 出品公司: | Ybscience |
|---|---|
| 载体名称: | pHIL-S1, pHIL S1 |
| 质粒类型: | 毕赤酵母蛋白表达载体 |
| 表达水平: | 高拷贝 |
| 启动子: | AOX1 |
| 克隆方法: | 多克隆位点，限制性内切酶 |
| 载体大小: | 8260 bp |
| 5' 测序引物及序列: | 5´ AOX1:5´-GACTGGTTCCAATTGACAAGC-3´； |
| 3' 测序引物及序列: | 3´ AOX1:5´-GCAAATGGCATTCTGACATCC-3´ |
| 载体标签: | N-PHO1分泌信号肽 |
| 载体抗性: | Ampicillin |
| 筛选标记: | HIS4 |
| 备注: | 利用PHO1信号肽，分泌表达蛋白。 |
| 稳定性: | 稳定 Stable |
| 组成型: | 组成型 Constitutive |
| 病毒/非病毒: | 非病毒 |
载体质粒图谱和多克隆位点信息
载体简介
pHIL-S1载体的详细信息如下：
• pHIL-S1载体大小8,260 bp，是融合表达载体。
• 载体构建过程中，可供使用的单一限制性内切酶位点为Xho I, EcoR I, Sma I, BamH I.
• 利用PHO1分泌信号肽，分泌表达蛋白基因。
• 在载体构建过程中，你的基因必须保证与信号肽的起始密码子的读码框一致。
• 毕赤酵母中利用HIS4进行筛选。
• 为了将基因插入GS115或KM71的AOX区, 使用SacI限制性内切酶线性化质粒(GS115中产生His+Mut+基因型，KM71中产生His+ MutS基因型)
•为了将基因插入GS115或KM71的His4区, 使用Sal I或者Stu I限制性内切酶线性化质粒(GS115中产生His+ Mut+基因型，KM71中产生His+ MutS基因型)
•将基因插入GS115的AOX1区域，需要使用Bgl II线性化质粒 (产生His+MutS基因型)。
载体序列
LOCUS pHIL-S1 8260 bp ds-DNA circular SYN 05-二月-2013
DEFINITION Pichia pastoris HIS4 vector for methanol-inducible expression of a
secreted protein.
ACCESSION .
VERSION .
KEYWORDS pHIL-S1
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT VNTAUTHORNAME|biofeng.com|
FEATURES Location/Qualifiers
source 1..8260
/organism="synthetic DNA construct"
/lab_host="Pichia pastoris"
/mol_type="other DNA"
promoter 2..941
/gene="Pichia pastoris AOX1"
/note="AOX1 promoter"
/note="inducible promoter, regulated by methanol"
/note="color: #ffffff; direction: RIGHT"
CDS 942..1007
/codon_start=1
/product="signal sequence from a secreted acid phosphatase
of Pichia pastoris"
/note="PHO1 signal sequence"
/note="synthetic gene fragment"
/note="color: #993366
Cleavage site after base 986"
/translation="MFSPILSLEIILALATLQSVFA"
misc_feature 1006..1026
/note="MCS"
/note="multiple cloning site"
/note="color: #99ccff"
terminator 1093..1339
/gene="Pichia pastoris AOX1"
/note="AOX1 terminator"
/note="transcription terminator for AOX1"
/note="color: #ffffff"
CDS complement(1752..4286)
/codon_start=1
/gene="Pichia pastoris HIS4"
/locus_tag="
"
/product="multifunctional enzyme, required for histidine
biosynthesis"
/note="PpHIS4"
/note="auxotrophic marker for Pichia pastoris"
/note="color: #ff7f50"
/protein_id="
"
/translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE
LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL
VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII
DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI
PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER
TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED
IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP
KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE
LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVETCP
GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE
VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA
LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED
AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV
QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK
HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"
misc_feature 4641..5397
/note="AOX1 3' fragment"
/note="region downstream of Pichia pastoris AOX1 gene"
/note="color: #ffffff"
rep_origin complement(5866..6454)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/note="color: #ffff00"
rep_origin 6565..7020
/direction=RIGHT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
/note="color: #ffff00"
CDS complement(7062..7922)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/note="This feature has 2 segments:
???1:?7062?..?7853?/?#ccffcc
???2:?7854?..?7922?/?#ccffcc?/?signal?sequence
Cleavage site after base 7853"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(7923..8027)
/gene="bla"
/note="AmpR promoter"
/note="color: #ffffff; direction: LEFT"
ORIGIN
1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag
61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt
121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc
181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta
241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggtcat gtttgtttat
301 ttccgaatgc aacaagctcc gcattacacc cgaacatcac tccagatgag ggctttctga
361 gtgtggggtc aaatagtttc atgttcccaa atggcccaaa actgacagtt taaacgctgt
421 cttggaacct aatatgacaa aagcgtgatc tcatccaaga tgaactaagt ttggttcgtt
481 gaaatgctaa cggccagttg gtcaaaaaga aacttccaaa agtcgccata ccgtttgtct
541 tgtttggtat tgattgacga atgctcaaaa ataatctcat taatgcttag cgcagtctct
601 ctatcgcttc tgaacccggt ggcacctgtg ccgaaacgca aatggggaaa caacccgctt
661 tttggatgat tatgcattgt cctccacatt gtatgcttcc aagattctgg tgggaatact
721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacaggcaa
781 tatataaaca gaaggaagct gccctgtctt aaaccttttt ttttatcatc attattagct
841 tactttcata attgcgactg gttccaattg acaagctttt gattttaacg acttttaacg
901 acaacttgag aagatcaaaa aacaactaat tattcgaaac gatgttctct ccaattttgt
961 ccttggaaat tattttagct ttggctactt tgcaatctgt cttcgctcga gaattccccg
1021 ggatccttag acatgactgt tcctcagttc aagttgggca cttacgagaa gaccggtctt
1081 gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt
1141 tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct
1201 tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg
1261 ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag
1321 tacagaagat taagtgagaa gttcgtttgt gcaagcttat cgataagctt taatgcggta
1381 gtttatcaca gttaaattgc taacgcagtc aggcaccgtg tatgaaatct aacaatgcgc
1441 tcatcgtcat cctcggcacc gtcaccctgg atgctgtagg cataggcttg gttatgccgg
1501 tactgccggg cctcttgcgg gatatcgtcc attccgacag catcgccagt cactatggcg
1561 tgctgctagc gctatatgcg ttgatgcaat ttctatgcgc acccgttctc ggagcactgt
1621 ccgaccgctt tggccgccgc ccagtcctgc tcgcttcgct acttggagcc actatcgact
1681 acgcgatcat ggcgaccaca cccgtcctgt ggatctatcg aatctaaatg taagttaaaa
1741 tctctaaata attaaataag tcccagtttc tccatacgaa ccttaacagc attgcggtga
1801 gcatctagac cttcaacagc agccagatcc atcactgctt ggccaatatg tttcagtccc
1861 tcaggagtta cgtcttgtga agtgatgaac ttctggaagg ttgcagtgtt aactccgctg
1921 tattgacggg catatccgta cgttggcaaa gtgtggttgg taccggagga gtaatctcca
1981 caactctctg gagagtaggc accaacaaac acagatccag cgtgttgtac ttgatcaaca
2041 taagaagaag cattctcgat ttgcaggatc aagtgttcag gagcgtactg attggacatt
2101 tccaaagcct gctcgtaggt tgcaaccgat agggttgtag agtgtgcaat acacttgcgt
2161 acaatttcaa cccttggcaa ctgcacagct tggttgtgaa cagcatcttc aattctggca
2221 agctccttgt ctgtcatatc gacagccaac agaatcacct gggaatcaat accatgttca
2281 gcttgagaca gaaggtctga ggcaacgaaa tctggatcag cgtatttatc agcaataact
2341 agaacttcag aaggcccagc aggcatgtca atactacaca gggctgatgt gtcattttga
2401 accatcatct tggcagcagt aacgaactgg tttcctggac caaatatttt gtcacactta
2461 ggaacagttt ctgttccgta agccatagca gctactgcct gggcgcctcc tgctagcacg
2521 atacacttag caccaacctt gtgggcaacg tagatgactt ctggggtaag ggtaccatcc
2581 ttcttaggtg gagatgcaaa aacaatttct ttgcaaccag caactttggc aggaacaccc
2641 agcatcaggg aagtggaagg cagaattgcg gttccaccag gaatatagag gccaactttc
2701 tcaataggtc ttgcaaaacg agagcagact acaccagggc aagtctcaac ttgcaacgtc
2761 tccgttagtt gagcttcatg gaatttcctg acgttatcta tagagagatc aatggctctc
2821 ttaacgttat ctggcaattg cataagttcc tctgggaaag gagcttctaa cacaggtgtc
2881 ttcaaagcga ctccatcaaa cttggcagtt agttctaaaa gggctttgtc accattttga
2941 cgaacattgt cgacaattgg tttgactaat tccataatct gttccgtttt ctggatagga
3001 cgacgaaggg catcttcaat ttcttgtgag gaggccttag aaacgtcaat tttgcacaat
3061 tcaatacgac cttcagaagg gacttcttta ggtttggatt cttctttagg ttgttccttg
3121 gtgtatcctg gcttggcatc tcctttcctt ctagtgacct ttagggactt catatccagg
3181 tttctctcca cctcgtccaa cgtcacaccg tacttggcac atctaactaa tgcaaaataa
3241 aataagtcag cacattccca ggctatatct tccttggatt tagcttctgc aagttcatca
3301 gcttcctccc taattttagc gttcaacaaa acttcgtcgt caaataaccg tttggtataa
3361 gaaccttctg gagcattgct cttacgatcc cacaaggtgg cttccatggc tctaagaccc
3421 tttgattggc caaaacagga agtgcgttcc aagtgacaga aaccaacacc tgtttgttca
3481 accacaaatt tcaagcagtc tccatcacaa tccaattcga tacccagcaa cttttgagtt
3541 gctccagatg tagcaccttt ataccacaaa ccgtgacgac gagattggta gactccagtt
3601 tgtgtcctta tagcctccgg aatagacttt ttggacgagt acaccaggcc caacgagtaa
3661 ttagaagagt cagccaccaa agtagtgaat agaccatcgg ggcggtcagt agtcaaagac
3721 gccaacaaaa tttcactgac agggaacttt ttgacatctt cagaaagttc gtattcagta
3781 gtcaattgcc gagcatcaat aatggggatt ataccagaag caacagtgga agtcacatct
3841 accaactttg cggtctcaga aaaagcataa acagttctac taccgccatt agtgaaactt
3901 ttcaaatcgc ccagtggaga agaaaaaggc acagcgatac tagcattagc gggcaaggat
3961 gcaactttat caaccagggt cctatagata accctagcgc ctgggatcat cctttggaca
4021 actctttctg ccaaatctag gtccaaaatc acttcattga taccattatt gtacaacttg
4081 agcaagttgt cgatcagctc ctcaaattgg tcctctgtaa cggatgactc aacttgcaca
4141 ttaacttgaa gctcagtcga ttgagtgaac ttgatcaggt tgtgcagctg gtcagcagca
4201 tagggaaaca cggcttttcc taccaaactc aaggaattat caaactctgc aacacttgcg
4261 tatgcaggta gcaagggaaa tgtcatactt gaagtcggac agtgagtgta gtcttgagaa
4321 attctgaagc cgtattttta ttatcagtga gtcagtcatc aggagatcct ctacgccgga
4381 cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac
4441 atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg
4501 ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca
4561 ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag
4621 gagtcgcata agggagagcg tcgagtatct atgattggaa gtatgggaat ggtgataccc
4681 gcattcttca gtgtcttgag gtctcctatc agattatgcc caactaaagc aaccggagga
4741 ggagatttca tggtaaattt ctctgacttt tggtcatcag tagactcgaa ctgtgagact
4801 atctcggtta tgacagcaga aatgtccttc ttggagacag taaatgaagt cccaccaata
4861 aagaaatcct tgttatcagg aacaaacttc ttgtttcgaa ctttttcggt gccttgaact
4921 ataaaatgta gagtggatat gtcgggtagg aatggagcgg gcaaatgctt accttctgga
4981 ccttcaagag gtatgtaggg tttgtagata ctgatgccaa cttcagtgac aacgttgcta
5041 tttcgttcaa accattccga atccagagaa atcaaagttg tttgtctact attgatccaa
5101 gccagtgcgg tcttgaaact gacaatagtg tgctcgtgtt ttgaggtcat ctttgtatga
5161 ataaatctag tctttgatct aaataatctt gacgagccaa ggcgataaat acccaaatct
5221 aaaactcttt taaaacgtta aaaggacaag tatgtctgcc tgtattaaac cccaaatcag
5281 ctcgtagtct gatcctcatc aacttgaggg gcactatctt gttttagaga aatttgcgga
5341 gatgcgatat cgagaaaaag gtacgctgat tttaaacgtg aaatttatct caagatctct
5401 gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
5461 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
5521 gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata
5581 ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga
5641 aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct
5701 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
5761 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
5821 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
5881 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
5941 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
6001 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
6061 atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
6121 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
6181 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
6241 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
6301 tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
6361 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
6421 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
6481 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
6541 aaggatcttc acctagatcc ttttacgcgc cctgtagcgg cgcattaagc gcggcgggtg
6601 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
6661 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
6721 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
6781 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
6841 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
6901 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
6961 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
7021 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
7081 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
7141 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
7201 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
7261 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
7321 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
7381 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
7441 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
7501 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
7561 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
7621 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
7681 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
7741 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
7801 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
7861 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
7921 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
7981 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
8041 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
8101 cgtatcacga ggccctttcg tcttcaagat ttctcatgtt tgacagctta tcatcgaatt
8161 aattctcatg tttgacagct tatcatcgat aagctgactc atgttggtat tgtgaaatag
8221 acgcagatcg ggaacactga aaaataacag ttattattcg
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digested plasmid (cut 5 µg of plasmid in volume of 20 µl) 51 µl labeled oligo 10 µl 10X Taq Buffer 6 µl 25 mM MgCl2 10 µl DMSO 2 µl 10 mM dNTP's 1 µl Taq polymerase 14 cycles of PCR Run on 6-8% polyacrylamide/urea gel Cut out probe and elute S1
相关专题 S1核酸酶 S1核酸酶来源于稻谷曲霉(Aspergillusoryzae)，是一种高度单链特异的核酸内切酶，在最适的酶催反应条件下，降解单链DNA或RNA，产生带5，-磷酸的单核苷酸或寡核苷酸。对双链DNA、双链RNA和DNA-RNA杂交体相对不敏感。通常水解单链DNA的速率要比水解双链DNA快75000倍。这种酶需要低水平的zn2+的激活，最适pH范围为4.0～4.3。一些螯合剂(如EDTA 和柠檬
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pHIL-S1
¥800 - 1200
