pPICZ A

pPICZ A

pPICZA载体基本信息

出品公司:	Ybscience
载体名称:	pPICZA, pPICZ A
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:	3329 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:	C-Myc, C-His
载体抗性:	Zeocin 博来霉素
筛选标记:	HIS4
备注:	插入基因是必需包含起始密码子ATG。
稳定性:	稳定 Stable
组成型:	组成型 Constitutive
病毒/非病毒:	非病毒

pPICZA载体质粒图谱和多克隆位点信息

pPICZA载体简介

pPICZ A，B和C是3.3 kb的毕赤酵母蛋白载体。表达的重组蛋白是融合蛋白，含有一个C-端多肽，多太重含c-myc和C-His标签。载体能够在毕赤酵母中利用甲醇诱导的高水平的表达目的蛋白，并且可以用在任何毕赤酵母中，包括X33，GS115菌株，SMD1168H，KM71H。

pPICZ系列载体包含以下元素：

•5'片段含有AOX1启动子的严格调控，利用甲醇诱导表达任何感兴趣的基因（Ellis等，1985; Koutz等人，1989;tschopp等人，1987A）。

•Zeocin抗性基因在大肠杆菌和毕赤酵母都能用于筛选（Baron等人,1992; Drocourt等人，1990）。

•C-端含Myc和His标签，可以用于检测和纯化重组蛋白。

•pPICZ A, B, C三种读码框使得可以将基因克隆入载体而不发生任何移码突变。

pPICZA载体序列

LOCUS       pPICZ A          3329 bp ds-DNA    circular SYN 
DEFINITION  Pichia pastoris vector for methanol-inducible intracellular 
            expression of a protein.
ACCESSION   .
VERSION     .
KEYWORDS    pPICZ A
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     Linearize with SacI, PmeI, or BstXI for integration at the AOX1 
            locus.
FEATURES             Location/Qualifiers
     source          1..3329
                     /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"
     misc_feature    932..1011
                     /note="MCS"
                     /note="multiple cloning site"
                     /note="color: #99ccff"
     CDS             1012..1041
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /note="Myc"
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             1057..1074
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     terminator      1153..1399
                     /gene="Pichia pastoris AOX1"
                     /note="AOX1 terminator"
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff"
     promoter        1439..1825
                     /gene="S. cerevisiae TEF1"
                     /note="TEF1 promoter"
                     /note="promoter for EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        1833..1880
                     /note="EM7 promoter"
                     /note="synthetic bacterial promoter?"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             1899..2273
                     /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      2339..2586
                     /gene="S. cerevisiae CYC1"
                     /note="CYC1 terminator"
                     /note="transcription terminator for CYC1"
                     /note="color: #ffffff"
     rep_origin      complement(2661..3249)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
                     /note="color: #ffff00"
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 aggaattcac gtggcccagc
      961 cggccgtctc ggatcggtac ctcgagccgc ggcggccgcc agcttgggcc cgaacaaaaa
     1021 ctcatctcag aagaggatct gaatagcgcc gtcgaccatc atcatcatca tcattgagtt
     1081 ttagccttag acatgactgt tcctcagttc aagttgggca cttacgagaa gaccggtctt
     1141 gctagattct aatcaagagg atgtcagaat gccatttgcc tgagagatgc aggcttcatt
     1201 tttgatactt ttttatttgt aacctatata gtataggatt ttttttgtca ttttgtttct
     1261 tctcgtacga gcttgctcct gatcagccta tctcgcagct gatgaatatc ttgtggtagg
     1321 ggtttgggaa aatcattcga gtttgatgtt tttcttggta tttcccactc ctcttcagag
     1381 tacagaagat taagtgagac cttcgtttgt gcggatcccc cacacaccat agcttcaaaa
     1441 tgtttctact ccttttttac tcttccagat tttctcggac tccgcgcatc gccgtaccac
     1501 ttcaaaacac ccaagcacag catactaaat tttccctctt tcttcctcta gggtgtcgtt
     1561 aattacccgt actaaaggtt tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg
     1621 tcgaaaaagg caataaaaat ttttatcacg tttctttttc ttgaaatttt tttttttagt
     1681 ttttttctct ttcagtgacc tccattgata tttaagttaa taaacggtct tcaatttctc
     1741 aagtttcagt ttcatttttc ttgttctatt acaacttttt ttacttcttg ttcattagaa
     1801 agaaagcata gcaatctaat ctaaggggcg gtgttgacaa ttaatcatcg gcatagtata
     1861 tcggcatagt ataatacgac aaggtgagga actaaaccat ggccaagttg accagtgccg
     1921 ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg
     1981 ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc
     2041 tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg
     2101 tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg
     2161 acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc
     2221 tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc
     2281 gacggcggcc cacgggtccc aggcctcgga gatccgtccc ccttttcctt tgtcgatatc
     2341 atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa
     2401 aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta
     2461 gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag acgcgtgtac
     2521 gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc gaaggcttta
     2581 atttgcaagc tggagaccaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2641 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2701 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2761 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2821 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt
     2881 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2941 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3001 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3061 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     3121 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3181 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3241 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3301 tcacgttaag ggattttggt catgagatc