pPICZα B

pPICZα B

pPICZalpha B载体基本信息

出品公司:	Ybscience
载体名称:	pPICZalpha B, pPICZα B, pPICZalphaB, pPICZαB
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:	3597 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

pPICZalpha B载体质粒图谱和多克隆位点信息

pPICZalpha B载体简介

pPICZα  A，B和C是3.6 kb的毕赤酵母蛋白分泌表达载体。表达的重组蛋白是融合蛋白，含有一个N-端多肽，编码酿酒酵母（Saccharomyces cerevisiae）α-因子分泌信号。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。

pPICZα  A，B和C系列载体包含以下元素：

•5'端含有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）。

•α-因子分泌信号能够分泌性表达目的蛋白。

•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。

pPICZalpha B载体序列

LOCUS       pPICZα B          3597 bp ds-DNA    circular SYN 05-二月-2013
DEFINITION  Pichia pastoris vector for methanol-inducible expression of a 
            secreted protein.
ACCESSION   .
VERSION     .
KEYWORDS    pPICZ(alpha) B
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..3597
                     /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"
     CDS             941..1207
                     /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:??941?..??997?/?#993366?/?presequence
                     ???2:??998?..?1195?/?#993366?/?pro?region
                     ???3:?1196?..?1207?/?#993366
                     Cleavage sites after bases 997, 1195"
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA"
     misc_feature    1206..1280
                     /note="MCS"
                     /note="multiple cloning site"
                     /note="color: #99ccff"
     CDS             1279..1308
                     /codon_start=1
                     /product="Myc (human c-Myc oncogene) epitope tag"
                     /note="Myc"
                     /note="color: #cc99b2"
                     /translation="EQKLISEEDL"
     CDS             1324..1341
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /note="color: #cc99b2"
                     /translation="HHHHHH"
     terminator      1421..1667
                     /gene="Pichia pastoris AOX1"
                     /note="AOX1 terminator"
                     /note="transcription terminator for AOX1"
                     /note="color: #ffffff"
     promoter        1707..2093
                     /gene="S. cerevisiae TEF1"
                     /note="TEF1 promoter"
                     /note="promoter for EF-1-alpha"
                     /note="color: #ffffff; direction: RIGHT"
     promoter        2101..2148
                     /note="EM7 promoter"
                     /note="synthetic bacterial promoter?"
                     /note="color: #ffffff; direction: RIGHT"
     CDS             2167..2541
                     /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      2607..2854
                     /gene="S. cerevisiae CYC1"
                     /note="CYC1 terminator"
                     /note="transcription terminator for CYC1"
                     /note="color: #ffffff"
     rep_origin      complement(2929..3517)
                     /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 atgagatttc cttcaatttt
      961 tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga
     1021 agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga
     1081 tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa
     1141 tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga aaagagaggc
     1201 tgaagctgca ggaattcacg tggcccagcc ggccgtctcg gatcggtacc tcgagccgcg
     1261 gcggccgcca gctttctaga acaaaaactc atctcagaag aggatctgaa tagcgccgtc
     1321 gaccatcatc atcatcatca ttgagtttgt agccttagac atgactgttc ctcagttcaa
     1381 gttgggcact tacgagaaga ccggtcttgc tagattctaa tcaagaggat gtcagaatgc
     1441 catttgcctg agagatgcag gcttcatttt tgatactttt ttatttgtaa cctatatagt
     1501 ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc ttgctcctga tcagcctatc
     1561 tcgcagctga tgaatatctt gtggtagggg tttgggaaaa tcattcgagt ttgatgtttt
     1621 tcttggtatt tcccactcct cttcagagta cagaagatta agtgagacct tcgtttgtgc
     1681 ggatccccca cacaccatag cttcaaaatg tttctactcc ttttttactc ttccagattt
     1741 tctcggactc cgcgcatcgc cgtaccactt caaaacaccc aagcacagca tactaaattt
     1801 tccctctttc ttcctctagg gtgtcgttaa ttacccgtac taaaggtttg gaaaagaaaa
     1861 aagagaccgc ctcgtttctt tttcttcgtc gaaaaaggca ataaaaattt ttatcacgtt
     1921 tctttttctt gaaatttttt tttttagttt ttttctcttt cagtgacctc cattgatatt
     1981 taagttaata aacggtcttc aatttctcaa gtttcagttt catttttctt gttctattac
     2041 aacttttttt acttcttgtt cattagaaag aaagcatagc aatctaatct aaggggcggt
     2101 gttgacaatt aatcatcggc atagtatatc ggcatagtat aatacgacaa ggtgaggaac
     2161 taaaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga
     2221 gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc
     2281 gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg
     2341 ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg
     2401 tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc
     2461 gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc
     2521 gtggccgagg agcaggactg acacgtccga cggcggccca cgggtcccag gcctcggaga
     2581 tccgtccccc ttttcctttg tcgatatcat gtaattagtt atgtcacgct tacattcacg
     2641 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt
     2701 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt
     2761 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga
     2821 gaaggttttg ggacgctcga aggctttaat ttgcaagctg gagaccaaca tgtgagcaaa
     2881 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2941 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3001 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3061 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3121 tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3181 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3241 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3301 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3361 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3421 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3481 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3541 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagatc