pGAPZα A

pGAPZα A

pGAPZalpha A载体基本信息

pGAPZalpha A载体质粒图谱和多克隆位点信息

pGAPZalpha A载体简介

甘油醛-3-磷酸脱氢酶（GAPDH）的酶在许多生物细胞中包括毕赤酵母，稳定高水平表达。 最近，GAPDH蛋白编码基因启动子（GAP）被深入研究，并在毕赤酵母中能够高水表达重组蛋白，这取决于使用的碳源（Waterham等人，1997）。GAP启动子（PGAP）的重组蛋白表达水平略高于AOX1启动子。

pGAPZ A，B，和C载体（2.9 KB）和pGAPZα，B，和C（3.1 KB）载体使用GAP启动子在毕赤酵母中稳定表达重组蛋白。重组蛋白表达为为带有C-末端myc抗原表位和C端His标签的融合蛋白。此外，pGAPZα 表达的融合蛋白在N-端带有酿酒酵母（Saccharomyces cerevisiae）的α-因子分泌信号肽。这两个系列的载体都提供三个不同读码框的载体，以方便克隆的带有C-端标签和/或N-末端分泌信号肽的载体 中。

这些载体是基于显性选择标记，Zeocin（博莱霉素）的筛选标记可以同时使用于毕赤酵母和大肠杆菌。

pGAPZalpha A载体序列

LOCUS       pGAPZα A          3147 bp ds-DNA    circular SYN 05-二月-2013
DEFINITION  Pichia pastoris vector for constitutive expression of a secreted 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    pGAPZ(alpha) A
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
COMMENT     This file is created by Vector NTI
COMMENT     Linearize within the GAP promoter for integration.
FEATURES             Location/Qualifiers
     source          1..3147
                     /organism="synthetic DNA construct"
                     /lab_host="Pichia pastoris"
                     /mol_type="other DNA"
     promoter        7..483
                     /note="GAP promoter"
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             493..759
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /note="alpha-factor secretion signal"
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence. The EA dipeptides are then removed by 
                     dipeptidyl aminopeptidase A."
                     /note="This feature has 3 segments:
                     ???1:?493?..?549?/?#993366?/?presequence
                     ???2:?550?..?747?/?#993366?/?pro?region
                     ???3:?748?..?759?/?#993366
                     Cleavage sites after bases 549, 747"
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA"
     misc_feature    760..828
                     /note="MCS"
                     /note="multiple cloning site"
                     /note="color: #99ccff"
     CDS             827..856
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /note="Myc"
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             872..889
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     terminator      968..1214
                     /gene="Pichia pastoris AOX1"
                     /note="AOX1 terminator"
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff"
     promoter        1229..1640
                     /gene="S. cerevisiae TEF1"
                     /note="TEF1 promoter"
                     /note="promoter for?EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        1647..1694
                     /note="EM7 promoter"
                     /note="synthetic bacterial promoter?"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             1713..2087
                     /codon_start=1
                     /gene="Sh?ble from?Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /note="BleoR"
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /note="color: #ccffcc"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     terminator      2153..2400
                     /gene="S. cerevisiae CYC1"
                     /note="CYC1 terminator"
                     /note="transcription terminator for CYC1"
                     /note="color: #ffffff"
     rep_origin      complement(2475..3063)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
ORIGIN
        1 agatcttttt tgtagaaatg tcttggtgtc ctcgtccaat caggtagcca tctctgaaat
       61 atctggctcc gttgcaactc cgaacgacct gctggcaacg taaaattctc cggggtaaaa
      121 cttaaatgtg gagtaatgga accagaaacg tctcttccct tctctctcct tccaccgccc
      181 gttaccgtcc ctaggaaatt ttactctgct ggagagcttc ttctacggcc cccttgcagc
      241 aatgctcttc ccagcattac gttgcgggta aaacggaggt cgtgtacccg acctagcagc
      301 ccagggatgg aaaagtcccg gccgtcgctg gcaataatag cgggcggacg catgtcatga
      361 gattattgga aaccaccaga atcgaatata aaaggcgaac acctttccca attttggttt
      421 ctcctgaccc aaagacttta aatttaattt atttgtccct atttcaatca attgaacaac
      481 tatttcgaaa cgatgagatt tccttcaatt tttactgctg ttttattcgc agcatcctcc
      541 gcattagctg ctccagtcaa cactacaaca gaagatgaaa cggcacaaat tccggctgaa
      601 gctgtcatcg gttactcaga tttagaaggg gatttcgatg ttgctgtttt gccattttcc
      661 aacagcacaa ataacgggtt attgtttata aatactacta ttgccagcat tgctgctaaa
      721 gaagaagggg tatctctcga gaaaagagag gctgaagctg aattcacgtg gcccagccgg
      781 ccgtctcgga tcggtacctc gagccgcggc ggccgccagc tttctagaac aaaaactcat
      841 ctcagaagag gatctgaata gcgccgtcga ccatcatcat catcatcatt gagttttagc
      901 cttagacatg actgttcctc agttcaagtt gggcacttac gagaagaccg gtcttgctag
      961 attctaatca agaggatgtc agaatgccat ttgcctgaga gatgcaggct tcatttttga
     1021 tactttttta tttgtaacct atatagtata ggattttttt tgtcattttg tttcttctcg
     1081 tacgagcttg ctcctgatca gcctatctcg cagctgatga atatcttgtg gtaggggttt
     1141 gggaaaatca ttcgagtttg atgtttttct tggtatttcc cactcctctt cagagtacag
     1201 aagattaagt gagaccttcg tttgtgcgga tcccccacac accatagctt caaaatgttt
     1261 ctactccttt tttactcttc cagattttct cggactccgc gcatcgccgt accacttcaa
     1321 aacacccaag cacagcatac taaattttcc ctctttcttc ctctagggtg tcgttaatta
     1381 cccgtactaa aggtttggaa aagaaaaaag agaccgcctc gtttcttttt cttcgtcgaa
     1441 aaaggcaata aaaattttta tcacgtttct ttttcttgaa attttttttt ttagtttttt
     1501 tctctttcag tgacctccat tgatatttaa gttaataaac ggtcttcaat ttctcaagtt
     1561 tcagtttcat ttttcttgtt ctattacaac tttttttact tcttgttcat tagaaagaaa
     1621 gcatagcaat ctaatctaag ggcggtgttg acaattaatc atcggcatag tatatcggca
     1681 tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg
     1741 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
     1801 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca
     1861 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg
     1921 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct
     1981 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg
     2041 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtccgacggc
     2101 ggcccacggg tcccaggcct cggagatccg tccccctttt cctttgtcga tatcatgtaa
     2161 ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac cgaaaaggaa
     2221 ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat gttagtatta
     2281 agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt gtacgcatgt
     2341 aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc tttaatttgc
     2401 aagctggaga ccaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2461 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2521 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2581 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2641 ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct cagttcggtg
     2701 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2761 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2821 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2881 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     2941 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3001 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3061 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3121 taagggattt tggtcatgca tgagatc
//

 pGAPZα A其他酵母表达载体：