pGAPZα B

pGAPZαB

pGAPZαB载体基本信息

pGAPZαB载体质粒图谱和多克隆位点信息

pGAPZαB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。

pGAPZα B载体序列

LOCUS       pGAPZα B           3151 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) B
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..3151
                     /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    758..832
                     /note="MCS"
                     /note="multiple cloning site"
                     /note="color: #99ccff"
     CDS             831..860
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /note="Myc"
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             876..893
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     terminator      972..1218
                     /gene="Pichia pastoris AOX1"
                     /note="AOX1 terminator"
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff"
     promoter        1233..1644
                     /gene="S. cerevisiae TEF1"
                     /note="TEF1 promoter"
                     /note="promoter for?EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        1651..1698
                     /note="EM7 promoter"
                     /note="synthetic bacterial promoter?"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             1717..2091
                     /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      2157..2404
                     /gene="S. cerevisiae CYC1"
                     /note="CYC1 terminator"
                     /note="transcription terminator for CYC1"
                     /note="color: #ffffff"
     rep_origin      complement(2479..3067)
                     /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 caggaattca cgtggcccag
      781 ccggccgtct cggatcggta cctcgagccg cggcggccgc cagctttcta gaacaaaaac
      841 tcatctcaga agaggatctg aatagcgccg tcgaccatca tcatcatcat cattgagttt
      901 tagccttaga catgactgtt cctcagttca agttgggcac ttacgagaag accggtcttg
      961 ctagattcta atcaagagga tgtcagaatg ccatttgcct gagagatgca ggcttcattt
     1021 ttgatacttt tttatttgta acctatatag tataggattt tttttgtcat tttgtttctt
     1081 ctcgtacgag cttgctcctg atcagcctat ctcgcagctg atgaatatct tgtggtaggg
     1141 gtttgggaaa atcattcgag tttgatgttt ttcttggtat ttcccactcc tcttcagagt
     1201 acagaagatt aagtgagacc ttcgtttgtg cggatccccc acacaccata gcttcaaaat
     1261 gtttctactc cttttttact cttccagatt ttctcggact ccgcgcatcg ccgtaccact
     1321 tcaaaacacc caagcacagc atactaaatt ttccctcttt cttcctctag ggtgtcgtta
     1381 attacccgta ctaaaggttt ggaaaagaaa aaagagaccg cctcgtttct ttttcttcgt
     1441 cgaaaaaggc aataaaaatt tttatcacgt ttctttttct tgaaattttt ttttttagtt
     1501 tttttctctt tcagtgacct ccattgatat ttaagttaat aaacggtctt caatttctca
     1561 agtttcagtt tcatttttct tgttctatta caactttttt tacttcttgt tcattagaaa
     1621 gaaagcatag caatctaatc taagggcggt gttgacaatt aatcatcggc atagtatatc
     1681 ggcatagtat aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt
     1741 ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg
     1801 ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg
     1861 ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg
     1921 cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac
     1981 gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg
     2041 cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtccga
     2101 cggcggccca cgggtcccag gcctcggaga tccgtccccc ttttcctttg tcgatatcat
     2161 gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa
     2221 ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt
     2281 attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc
     2341 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat
     2401 ttgcaagctg gagaccaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     2461 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     2521 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     2581 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     2641 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
     2701 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     2761 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     2821 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     2881 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     2941 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3001 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3061 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     3121 acgttaaggg attttggtca tgcatgagat c
//

其他酵母表达载体：