| 出品公司: | Invitrogen |
|---|---|
| 载体名称: | pAO815 |
| 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: | 7709 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: | / |
| 载体抗性: | Ampicillin |
| 筛选标记: | HIS4 |
| 备注: |
插入基因是必需包含起始密码子ATG。
|
| 产品目录号: | K173-20 |
| 稳定性: | 稳定 Stable |
| 组成型: | 组成型 Constitutive |
| 病毒/非病毒: | 非病毒 |
买家导航
pAO815载体质粒基本信息
pAO815质粒图谱载体图谱和pAO815载体序列质粒序列多克隆位点信息
pAO815质粒载体简介
pAO815是一个鉴定多拷贝基因插入到毕赤酵母基因组中的载体。
1. pAO815载体大小为7709 bp。
2. 在多克隆位点区域，拥有1个单一的限制性酶切位点EcoR I。
3.胞内表达。
4.载体构建时，需要包含一个ATG启动子，同时需要加入Kozak序列。 (Cavener and Stuart, 1991; Kozak, 1987; Kozak, 1990)
5. 毕赤酵母中使用HIS4基因筛选。
6. 将基因插入GS115和KM71的HIS4中，需要使用Sal I或者Stu I线性化质粒(GS115产生的基因型为His+ Mut+，KM71酵母产生的基因型为His+ MutS)。
7. 将基因插入GS115的AOX1基因中，也可以使用Bgl II 限制酶线性化质粒，产生的酵母基因型为His+ MutS。
pAO815质粒序列载体序列
LOCUS pAO815 7709 bp ds-DNA circular SYN 05-二月-2013
DEFINITION Pichia pastoris?vector for creating multimers to drive high levels
of methanol-inducible protein expression.
ACCESSION .
VERSION .
KEYWORDS pAO815
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
COMMENT This file is created by Vector NTI
http://www.biofeng.com/
COMMENT VNTAUTHORNAME|biofeng.com|
COMMENT The gene of interest can be inserted at the unique EcoRI site.
Multimers of the resulting BglII-BamHI expression cassette can be
inserted at the BamHI site.
FEATURES Location/Qualifiers
source 1..7709
/organism="synthetic DNA construct"
/lab_host="Pichia pastoris"
/mol_type="other DNA"
promoter 2..940
/gene="Pichia pastoris AOX1"
/note="AOX1 promoter"
/note="inducible promoter, regulated by methanol"
/note="color: #ffffff; direction: RIGHT"
terminator 1018..1264
/gene="Pichia pastoris AOX1"
/note="AOX1 terminator"
/note="transcription terminator for AOX1"
/note="color: #ffffff"
CDS complement(1665..4199)
/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 4554..5310
/note="AOX1 3' fragment"
/note="region downstream of Pichia pastoris AOX1 gene"
/note="color: #ffffff"
rep_origin complement(5780..6368)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/note="color: #ffff00"
CDS complement(6539..7399)
/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:?6539?..?7330?/?#ccffcc
???2:?7331?..?7399?/?#ccffcc?/?signal?sequence
Cleavage site after base 7330"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(7400..7504)
/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 gcgaggttca tgtttgttta
301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt
541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
901 caacttgaga agatcaaaaa acaactaatt attcgaaacg aggaattcgc cttagacatg
961 actgttcctc agttcaagtt gggcacttac gagaagaccg gtcttgctag attctaatca
1021 agaggatgtc agaatgccat ttgcctgaga gatgcaggct tcatttttga tactttttta
1081 tttgtaacct atatagtata ggattttttt tgtcattttg tttcttctcg tacgagcttg
1141 ctcctgatca gcctatctcg cagctgatga atatcttgtg gtaggggttt gggaaaatca
1201 ttcgagtttg atgtttttct tggtatttcc cactcctctt cagagtacag aagattaagt
1261 gagacgttcg tttgtgcgga tcctaatgcg gtagtttatc acagttaaat tgctaacgca
1321 gtcaggcacc gtgtatgaaa tctaacaatg cgctcatcgt catcctcggc accgtcaccc
1381 tggatgctgt aggcataggc ttggttatgc cggtactgcc gggcctcttg cgggatatcg
1441 tccattccga cagcatcgcc agtcactatg gcgtgctgct agcgctatat gcgttgatgc
1501 aatttctatg cgcacccgtt ctcggagcac tgtccgaccg ctttggccgc cgcccagtcc
1561 tgctcgcttc gctacttgga gccactatcg actacgcgat catggcgacc acacccgtcc
1621 tgtggatcta tcgaatctaa atgtaagtta aaatctctaa ataattaaat aagtcccagt
1681 ttctccatac gaaccttaac agcattgcgg tgagcatcta gaccttcaac agcagccaga
1741 tccatcactg cttggccaat atgtttcagt ccctcaggag ttacgtcttg tgaagtgatg
1801 aacttctgga aggttgcagt gttaactccg ctgtattgac gggcatatcc gtacgttggc
1861 aaagtgtggt tggtaccgga ggagtaatct ccacaactct ctggagagta ggcaccaaca
1921 aacacagatc cagcgtgttg tacttgatca acataagaag aagcattctc gatttgcagg
1981 atcaagtgtt caggagcgta ctgattggac atttccaaag cctgctcgta ggttgcaacc
2041 gatagggttg tagagtgtgc aatacacttg cgtacaattt caacccttgg caactgcaca
2101 gcttggttgt gaacagcatc ttcaattctg gcaagctcct tgtctgtcat atcgacagcc
2161 aacagaatca cctgggaatc aataccatgt tcagcttgag acagaaggtc tgaggcaacg
2221 aaatctggat cagcgtattt atcagcaata actagaactt cagaaggccc agcaggcatg
2281 tcaatactac acagggctga tgtgtcattt tgaaccatca tcttggcagc agtaacgaac
2341 tggtttcctg gaccaaatat tttgtcacac ttaggaacag tttctgttcc gtaagccata
2401 gcagctactg cctgggcgcc tcctgctagc acgatacact tagcaccaac cttgtgggca
2461 acgtagatga cttctggggt aagggtacca tccttcttag gtggagatgc aaaaacaatt
2521 tctttgcaac cagcaacttt ggcaggaaca cccagcatca gggaagtgga aggcagaatt
2581 gcggttccac caggaatata gaggccaact ttctcaatag gtcttgcaaa acgagagcag
2641 actacaccag ggcaagtctc aacttgcaac gtctccgtta gttgagcttc atggaatttc
2701 ctgacgttat ctatagagag atcaatggct ctcttaacgt tatctggcaa ttgcataagt
2761 tcctctggga aaggagcttc taacacaggt gtcttcaaag cgactccatc aaacttggca
2821 gttagttcta aaagggcttt gtcaccattt tgacgaacat tgtcgacaat tggtttgact
2881 aattccataa tctgttccgt tttctggata ggacgacgaa gggcatcttc aatttcttgt
2941 gaggaggcct tagaaacgtc aattttgcac aattcaatac gaccttcaga agggacttct
3001 ttaggtttgg attcttcttt aggttgttcc ttggtgtatc ctggcttggc atctcctttc
3061 cttctagtga cctttaggga cttcatatcc aggtttctct ccacctcgtc caacgtcaca
3121 ccgtacttgg cacatctaac taatgcaaaa taaaataagt cagcacattc ccaggctata
3181 tcttccttgg atttagcttc tgcaagttca tcagcttcct ccctaatttt agcgttcaac
3241 aaaacttcgt cgtcaaataa ccgtttggta taagaacctt ctggagcatt gctcttacga
3301 tcccacaagg tggcttccat ggctctaaga ccctttgatt ggccaaaaca ggaagtgcgt
3361 tccaagtgac agaaaccaac acctgtttgt tcaaccacaa atttcaagca gtctccatca
3421 caatccaatt cgatacccag caacttttga gttgctccag atgtagcacc tttataccac
3481 aaaccgtgac gacgagattg gtagactcca gtttgtgtcc ttatagcctc cggaatagac
3541 tttttggacg agtacaccag gcccaacgag taattagaag agtcagccac caaagtagtg
3601 aatagaccat cggggcggtc agtagtcaaa gacgccaaca aaatttcact gacagggaac
3661 tttttgacat cttcagaaag ttcgtattca gtagtcaatt gccgagcatc aataatgggg
3721 attataccag aagcaacagt ggaagtcaca tctaccaact ttgcggtctc agaaaaagca
3781 taaacagttc tactaccgcc attagtgaaa cttttcaaat cgcccagtgg agaagaaaaa
3841 ggcacagcga tactagcatt agcgggcaag gatgcaactt tatcaaccag ggtcctatag
3901 ataaccctag cgcctgggat catcctttgg acaactcttt ctgccaaatc taggtccaaa
3961 atcacttcat tgataccatt attgtacaac ttgagcaagt tgtcgatcag ctcctcaaat
4021 tggtcctctg taacggatga ctcaacttgc acattaactt gaagctcagt cgattgagtg
4081 aacttgatca ggttgtgcag ctggtcagca gcatagggaa acacggcttt tcctaccaaa
4141 ctcaaggaat tatcaaactc tgcaacactt gcgtatgcag gtagcaaggg aaatgtcata
4201 cttgaagtcg gacagtgagt gtagtcttga gaaattctga agccgtattt ttattatcag
4261 tgagtcagtc atcaggagat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
4321 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
4381 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
4441 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
4501 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagta
4561 tctatgattg gaagtatggg aatggtgata cccgcattct tcagtgtctt gaggtctcct
4621 atcagattat gcccaactaa agcaaccgga ggaggagatt tcatggtaaa tttctctgac
4681 ttttggtcat cagtagactc gaactgtgag actatctcgg ttatgacagc agaaatgtcc
4741 ttcttggaga cagtaaatga agtcccacca ataaagaaat ccttgttatc aggaacaaac
4801 ttcttgtttc gaactttttc ggtgccttga actataaaat gtagagtgga tatgtcgggt
4861 aggaatggag cgggcaaatg cttaccttct ggaccttcaa gaggtatgta gggtttgtag
4921 atactgatgc caacttcagt gacaacgttg ctatttcgtt caaaccattc cgaatccaga
4981 gaaatcaaag ttgtttgtct actattgatc caagccagtg cggtcttgaa actgacaata
5041 gtgtgctcgt gttttgaggt catctttgta tgaataaatc tagtctttga tctaaataat
5101 cttgacgagc caaggcgata aatacccaaa tctaaaactc ttttaaaacg ttaaaaggac
5161 aagtatgtct gcctgtatta aaccccaaat cagctcgtag tctgatcctc atcaacttga
5221 ggggcactat cttgttttag agaaatttgc ggagatgcga tatcgagaaa aaggtacgct
5281 gattttaaac gtgaaattta tctcaagatc tgctgcctcg cgcgtttcgg tgatgacggt
5341 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
5401 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
5461 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
5521 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
5581 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
5641 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
5701 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
5761 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
5821 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
5881 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
5941 cctttctccc ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt
6001 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
6061 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
6121 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
6181 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
6241 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
6301 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
6361 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
6421 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
6481 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
6541 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
6601 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
6661 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
6721 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
6781 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
6841 ttgttgccat tgctgcaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
6901 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
6961 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
7021 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
7081 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
7141 cttgcccggc gtcaacacgg gataataccg cgccacatag cagaacttta aaagtgctca
7201 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
7261 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
7321 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
7381 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
7441 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
7501 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
7561 taacctataa aaataggcgt atcacgaggc cctttcgtct tcaagaatta attctcatgt
7621 ttgacagctt atcatcgata agctgactca tgttggtatt gtgaaataga cgcagatcgg
7681 gaacactgaa aaataacagt tattattcg
//
