胞外表达载体,pIC9K m or U山 Sac I AOX1(TT) Bgl l Comments for pPIC9K: 9276 nucleotides 5 AOXI promoter fragment: bases 1-948 5AOXI primer site: bases 855-875 a-Factor secretion signal(s): bases 949-1218 Sal a-Factor primer site: bases 1152-1172 Multiple Cloning Site: bases 1192-1241 pPIC9K 3AOXI primer site: bases 1327-1347 3/ transcription 9. 3 kb termination (TT): bases 1253-1586 HIS4 ORF: bases 4514-1980 Kanamycin resistance gene: bases 5743-4928 Bgl ll 3 AOXI fragment: bases 6122-6879 ColEl origin: bases 7961-7288 Ampicillin resistance gene: bases 8966-8106 Kanam
11 胞外表达载体, pIC9K
AOXI mRNA 5'end(824) 5.OX/ prmer site (855-875 82 TTATCATCAT TATTAGCTTA CTTTCATAAT TGCGACTGGT TCCAATTGAC 87 AAGCTTTTGA TTTTAACGAC TTTTAACGAC AACTTGAGAA GATCAAAAAA 2< pPIC9k,胞外表达载体 ACG ATG AGA"TsTr 96 AC GCA GTTA 1宁CGeT己 TCC GCATT GO宁GcT Phe Thr Ala val Leu phe Ala Ala ser ser Ala leu Ala Ala 100 CCA GTC AAC ACT ACA ACA GAA GAT GAA ACG GCA CAA ATT CCG Pro Val Asn ThrThrThr Glu Asp Glu Thr Ala Gln Ile Pro 105 GCT GAA GCT GTC ATC GGT TAC TCA GAT TTA GAA GGG GAT TTC Ala Glu Ala val Ile Gly Tyr ser Asp Leu Glu Gly Asp Phe 109 GAT GTT GCT GTT TTG CCATTT TCC AAC AGC ACA AAT GGG Asp Val Ala val Leu ro phe ser Asn ser Thr AsnAsn\Gly a-Factor prmer site (1 152-1172) 113 TTA TTG TTT ATA AATACT ACT ATT GCC Leu Leu phe Ile Asn rThr工1eA ex工1eA1aA1 Kex2 signal c 117 GAA GGG GTA TCT GAG AA AGA GAG GCT GAA GCT TAC Iu Glu Gly Val ser le Arg Glu AIaiGlu Alatyr Eco RI Nor I Ste 13 signal cleavage 121GTA GAA TTC CCT AGG GCG G GCGAAT TAA TTCGCCTTAG Glu phe pro Arg Alala Ala ★★ 125 ACATGACTGT TCCTCAGTTC AAGTTGGGCA CTTACGAGAA GACCGGTCTT 3'4OX 1 primer site(1327-1347) 130 GCTAGATTCT AATCAAGAGG ATGTCAGAAT GCCATTTGCC TGAGAGATGC 135 AGGCTTCATT TTTGATACTT TTTTATTTGT AACCTATATA GTATAGGATT ↓ AOXI mRNA3end(1418) 140 TTTTTTGTCA
12 pPIC9k, 胞外表达载体
3.多拷贝产生,pIC9K,体内形成多拷贝 了A0Mn:{p"№nMHA Expression Cassette 1 2nd insertion Event Expression Cassette 2 3rd Insertion Event, etc. 未线性化的环状质粒发生同源重组的几率非常低
13 3. 多拷贝产生，pIC9K, 体内形成多拷贝 因为未线性化的环状质粒之间发生同源 重组的几率非常低 未线性化的环状质粒发生同源 重组的几率非常低
pAO815 可体外构建多拷贝 3 AOX1(TT) 5 AOX1 BamH Sal pAo815 Stu I 7.7kb AOX1 Bgl ll 14
14 pAO815 可体外构建多拷贝