pPIC9K序列.docx
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pPIC9K序列
载体基本信息
出品公司:
载体名称:
质粒类型:
InvitrogenpPIC9K,pPIC9K毕赤酵母蛋口表达载体
表达水平:
启动子:
高拷贝
AOX1
克隆方法:
载体大小:
5,测序引物及序列:
3'测序引物及序列:
载体标签:
多克隆位点,限制性内切酶
9276bp
5'AOX1:
5,-GACTGGTTCCAATTGACAAGC-3,;
alpha—Factor-F:
5,一TACTATTGCCAGCATTGCTGC—3'
3'A0X1:
5'—GCAAATGGCATTCTGACATCC—3'
N-alphafactor
载体抗性:
氨节和卡那
筛选标记:
备注:
HIS4
利用alpha分泌因子,分泌表达蛋白。
产品目录号:
V175-20
稳定性:
组成型:
稳定Stable
组成型Constitutive
病毒/非病毒:
非病毒
-i
=1一¥山一meus
BglW
3,A0X1(TT)
pPIC9KI
9-3kb*
BglW
Kan回
PA0X1andMultipleCloningSiteofpPIC9K
Thesequencebelowshowsthedetailofthemultiplecloningsiteandsurroundingsequences・
5’以7pnmersite门55・*75)
CTTTCATAATTGCGACTGGTTCCAATTGAC
AOXimRNA3d(824)
ttItcatcattattagctta
AAGC*TTTTGATTTTAACGACTTTTAACGACAACTTGAGAAGATCAAAAAAStart(949)a-FactorSignalSequenceCAACTAATTATTCGAAGGATCCAAACGATGAGATTTCCTTCAATTMetArgPheProSerTie
TTT
ACT
GCA
GTT
TTA
TTC
GCA
GCA
TCC
TCC
GCA
TTA
GCT
GCT
Phe
Thr
Ala
Vai
Leu
Phe
Ala
Ala
Ser
Ser
Ala
Leu
Ala
Ala
CCA
GTC
AAC
ACT
ACA
ACA
GAA
GAT
GAA
ACG
GCA
CAA
ATT
CCG
Pro
Vai
Asn
Thr
Thr
Thr
G1U
Asp
Glu
Thr
Ala
Gin
Tie
Pro
GCT
GAA
GCT
GTC
ATC
GGT
TAC
TCA
GAT
TTA
GAA
GGG
GAT
TTC
Ala
Glv
Ala
Vai
He
Gly
Tyr
Ser
Asp
Leu
Glu
Gly
Asp
Phe
GAT
GTT
GCT
GTT
TTG
CCA
TTT
TCC
AAC
AGC
ACA
AAT
AAC
GGG
Asp
Vai
Ala
Vai
Leu
Pro
Phe
Ser
Asn
Ser
Thr
Asn
Asn
Gly
CL・Faci(〉rprimer(1152・1172)
TTA
TTG
TTT
ATA
AAT
ACT
ACT
ATT
GCC
AGC
ATT
GCT
GCT
AAA
Leu
Leu
Phe
He
Asn
Thr
Thr
lie
Ala
Ser
He
Ala
Ala
Lys
VSignalcleavage(1203-1204)曲B,
GAA
GAA
GGG
GTA
TCT
CTC
GAG
AAA
AGA
GAG
GCT
GAA
GCT
TAC
Glu
Glu
Gly
Vai
Ser
Leu
Glu
Lys
Arg
Glu
Ala
Glu
Ala
Tyr
EgRl
AvrII
No\1
GTA
GAA
TTC
CCT
AGG
GCG
GCC
GCG
AAT
TAA
TTCGCCTTAG
Vai
Glu
Phe
Pro
Arg
Ala
Ala
Ala
Asn
***
ACATGACTGTTCCTCAGTTCAAGTTGGGCACTTACGAGAAGACCGGTCTT
3*AOXIprimersite(1327-1347)
GCTAGATTCTAATCAkdkGGATGTCAGAATGCCATTTGCC*TGAGAGATGC
AGGCTTCATTTTTGATACTTTTTTATTTGTAACCTATATAGTATAGGATT
JA()X\mRNA3'cnd(141X)
TTTTTTGTCA
载体简介
PPIC9K载体中存在卡那抗性基因,允许利用卡那抗性在酵母体内筛选多克隆拷贝,载体其余部分与
PPIC9载体完全一样。
PPIC9K载体能够使用的毕赤酵母宿主菌是KM71和GS115=
PPIC9K载体详细信息如卜•:
•PPIC9载体大小9276bp,是融合表达载体。
•载体构建过程中,町供使用的单一限制性内切酸位点为SnaBLEcoRI.AvrIIZNotI.
•利用alpha因子分泌信号肽,分泌表达蛋白基因。
・在载体构建过程中,你的基因必须保证与信号肽的起始密码子的读码框一致。
・毕赤酵母中利用HIS4进行筛选.
・为了将基因插入GS115或KM71的AOX区,使用SacI限制性内切酸线性化质粒(GS115中产生His+Mut+基因型,KM71中产生His+MutS基因型)
•为了将基因插入GSU5或KM71的His4区,使用SalI或者StuI限制性内切酸线性化质純
(GS115中产生His+Mut+基因型,KM71中产生His+MutS基因型)
•将基因插入GS115的AOX1区域,需要使用BglII线性化质粒(产生His+MutS基因型).
载体序列
LOCUSpPIC9K9276bpDMAcircularSYN
DEFINITIONpPICSK
ACCESSION
KEYWORDS
SOURCE
ORGANISM
COMMENT
COMMENT
othersequences;artificialsequences:
vectors,
ThiscreatedbyVectorNTI
VNTAUTEORNAMEIbiofeng.coml
source
1..9276
/crganism=,,pPIC9Kzz
/mol_type=,rotherDNA,f
/label=Malpha_factor_primerM
miscfeature
complement(1327.61347)
CDS
complement(1980。
■4514)
/label=/zORFframe2M
gene
complement(4928..5743)
/label=MKanR2z,
/gene=,zKanR2r,
CDS
complement(4928..5743)
/label=zzORFframe3"
misc_feature
complement(5908..5927)
/label=MpBRrevBam_primer,z
CDS
complement(5909-.6631〉
/label=zzORFframe3”
misc_feacure
complement(6913.<>6935)
/label=MpGEX_3_primer,z
rep_origin
complement(7332..7951)
/label=zzpBR322_originzz
gene
complement(8106..8966)
/label=z,AirtpicillinM
/gene=,zAmpicillinM
CDS
complement(8106.•8966)
/label=MORFframe2M
promoter
complement(9008.-9036)
/label=〃AmpR_prcmocer"
ORIGIN
1AGATCTAACATCCAAAGACGAAAGGTTGAATGAAACCTTTTTGCCATCCGACATCCACAG
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
CGCAT1ACAC
CCGAACATCA
CTCCAGATGA
GGGCTTTCTG
361
AGTGTGGGGT
CAAATAGTTT
CAIGTTCCCC
AAATGGCCCA
AAACTGACAG
TTTAAACGCT
421
GTCTTGGAAC
CTAATATGAC
AAAAGCGTGA
TCTCATCCAA
GATGAACTAA
GTTTGGTTCG
481
TTGAAATGCT
AACGGCCAGT
TGGTCAAAAA
GAAACTTCCA
AAAGTCGCCA
TACCGTTTGT
541
CTTGTTTGGT
ATTGATTGAC
GAATGCTCAA
AAATAATCTC
ATTAATGCTT
AGCGCAGTCT
601
CTCTATCGCT
TCTGAACCCC
GGTGCACCTG
TGCCGAAACG
CAAAIGGGGA
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
ATTCGAAGGA
TCCAAACGAT
GAGATTTCCT
961
TCAATTTTTA
CTGCAGTTTT
ATTCGCAGCA
TCCTCCGCAT
TAGCTGCTCC
AGTCAACACT
1021
ACAACAGAAG
ATGAAACGGC
ACAAATTCCG
GCTGAAGCTG
TCATCGGTTA
CTCAGATTTA
1081
GAAGGGGATT
TCGATGTTGC
TGTTTTGCCA
TTTTCCAACA
GCACAAATAA
CGGGTTATTG
1141
TTTATAAATA
CTACTATTGC
CAGCATTGCT
GCTAAAGAAG
AAGGGGTATC
TCTCGAGAAA
1201
AGAGAGGCTG
AAGCTTACGT
AGAATTCCCT
AGGGCGGCCG
CGAATTAATT
CGCCTTAGAC
1261
ATGACTG1TC
CTCAGT1CAA
GTTGGGCACT
TACGAGAAGA
CCGGTCTTGC
TAGATTCTAA
1321
TCAAGAGGAT
GTCAGAATGC
CATT1GCCTG
AGAGATGCAG
GCTTCATTTT
TGATACTTTT
1381
TTATTTGTAA
CCTATATAGT
ATAGGATTTT
TTTTGTCATT
TTGTTTCTTC
TCGTACGAGC
1441
TTGCTCCTGA
TCAGCCTATC
TCGCAGCTGA
TGAATATCTT
GTGGTAGGGG
TTTGGGAAAA
1501
TCATTCGAGT
TTGATGTTTT
TCTTGGTATT
TCCCACTCCT
CTTCAGAGTA
CAGAAGATTA
1561
1621
1681
1741
1801
1861
1921
1981
2041
2101
2161
2221
2281
2341
2401
2461
2521
2581
2641
2701
2761
2821
2881
2941
3001
pP1C9K序列
AGTGAGAAGTTCGTTTGTGC
AAGC1TATCGATAAGCTTTAATGCGGTAGTTTATCACAGT
TAAATTGCTAACGCAGTCAG
GCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCC
TCGGCACCGTCACCCTGGAT
GCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCC
TCTTGCGGGATATCGTCCAT
TCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGC
TATATGCGTTGATGCAATTT
CTATGCGCACCCGTICTCGGAGCACTGTCCGACCGCTTTG
GCCGCCGCCCAGTCCTGCTC
GCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGG
CGACCACACCCGTCCTGTGG
ATCTATCGAATCTAAATGTAAGTTAAAATCTCTAAATAAT
TAAATAAGTCCCAGTTTCTC
CATACGAACCTTAACAGCATTGCGGTGAGCATCTAGACCT
TCAACAGCAGCCAGATCCAT
CACTGCTTGGCCAATATGTTTCAGTCCCTCAGGAGT1ACG
TCTTGTGAAGTGATGAACTT
CTGGAAGGTTGCAGTGTTAACTCCGCTGTATTGACGGGCA
TATCCGTACGTTGGCAAAGT
GTGGTTGGTACCGGAGGAGTAATCTCCACAACTCTCTGGA
GAGTAGGCACCAACAAACAC
AGATCCAGCGTGTTGTACTTGATCAACATAAGAAGAAGCA
TTCTCGATTTGCAGGATCAA
GTGTTCAGGAGCGTACTGAITGGACATTTCCAAAGCCTGC
TCGTAGGTTGCAACCGATAG
GGTTGTAGAGTGTGCAATACACTTGCGTACAATITCAACC
CT1GGCAACTGCACAGCTTG
GTTGTGAACAGCATCTTCAATTCTGGCAAGCTCCTTGTCT
GTCATATCGACAGCCAACAG
AATCACCTGGGAATCAATACCATGTTCAGCT1GAGACAGA
AGGTCTGAGGCAACGAAATC
TGGATCAGCGTATTTATCAGCAATAACTAGAACTTCAGAA
GGCCCAGCAGGCATGTCAAT
ACTACACAGGGCTGATGTGTCAITTTGAACCATCATCTTG
GCAGCAGTAAOGAACTGGTT
TCCTGGACCAAATAITTTGTCACACTTAGGAACAGTTTCT
GTTCCGTAAGCCATAGCAGC
TACTGCCTGGGCGCCTCCTGCTAGCACGATACACTTAGCA
CCAACCTTGTGGGCAACGTA
GATGACTTCTGGGGTAAGGGTACCATCCTTCTTAGGTGGA
GATGCAAAAACAATTTCTTT
GCAACCAGCAACTTTGGCAGGAACACCCAGCATCAGGGAA
GTGGAAGGCAGAATTGCGGT
TCCACCAGGAATATAGAGGCCAACTTTCTCAATAGG1CTT
GCAAAACGAGAGCAGACTAC
ACCAGGGCAAGTCTCAACTTGCAACGTCTCCGTTAGTTGA
GCTTCATGGAATTTCCTGAC
GTTATCTATAGAGAGATCAATGGCTCTCTTAACGTTATCT
3061GGCAATTGCATAAGTTCCTC
TGGGAAAGGAGCTTCTAACACAGGTGTCTT
CAAAGCGACT
3121CCATCAAACTTGGCAGTTAG
TTCTAAAAGGGCTTTGTCACCATTTTGACG
AACATTGTCG
3181ACAATTGGTTTGACTAATTC
CATAATCTGTTCCGTTTTCIGGATAGGACG
ACGAAGGGCA
3241TCTTCAATTTCTTGTGAGGA
GGCCTTAGAAACGTCAATTTTGCACAATTC
AATACGACCT
3301TCAGAAGGGACTTCTTTAGG
TTTGGATTCTTCTTTAGGTTGTTCCTTGGT
GTATCCTGGC
3361TTGGCATCTCCTTTCCTTCT
AGTGACCTTTAGGGACTTCATATCCAGGTT
TCTCTCCACC
3421TCGTCCAACGTCACACCGTA
CTTGGCACATCTAACTAATGCAAAATAAAA
TAAGTCAGCA
3431CATTCCCAGGCTATATCTTC
CTTGGATTTAGCTTCTGCAAGTTCATCAGC
TTCCTCCCTA
3541ATTTTAGCGTTCAACAAAAC
TTCGTCGTCAAATAACCGTTTGGTATAAGA
ACCTTCTGGA
3601GCATTGCTCTTACGATCCCA
CAAGGTGGCTTCCATGGCTCTAAGACCCTT
TGATTGGCCA
3661AAACAGGAAGTGCGTTCCAA
GTGACAGAAACCAACACC1GTTTGTTCAAC
CACAAATTTC
3721AAGCAGTCTCCATCACAATC
CAATTCGATACCCAGCAACTTTTGAGTTGC
TCCAGATGTA
3781GCACCTTTATACCACAAACC
GTGACGACGAGATTGGTAGACTCCAGTTTG
TGTCCTTATA
3841GCCTCCGGAATAGACTTTTT
GGACGAGTACACCAGGCCCAACGAGTAATT
AGAAGAGTCA
3901GCCACCAAAGTAGTGAATAG
ACCATCGGGGCGGTCAGTAGTCAAAGACGC
CAACAAAATT
3961TCACTGACAGGGAACTTTTT
GACATCTTCAGAAAGTTCGTATTCAGTAGT
CAATTGCCGA
4021GCATCAAIAATGGGGATTAT
ACCAGAAGCAACAGTGGAAGTCACATCTAC
CAACTTTGCG
4031GTCTCAGAAAAAGCATAAAC
AGTTCTACTACCGCCATTAGTGAAACTTTT
CAAATCGCCC
4141AGTGGAGAAGAAAAAGGCAC
AGCGATACTAGCATTAGCGGGCAAGGATGC
AACTTTATCA
4201ACCAGGG1CCTATAGATAAC
CCTAGCGCCTGGGATCATCCTTTGGACAAC
TCTTTCTGCC
4261AAATCTAGGTCCAAAATCAC
TTCATTGATACCAT7ATTGTACAACTTGAG
CAAGTTGTCG
4321ATCAGCTCCTCAAATTGGTC
CTCTGTAACGGATGACTCAACTTGCACATT
AACTTGAAGC
4381TCAGTCGATTGAGTGAACTT
GATCAGGTTGTGCAGCTGGTCAGCAGCATA
GGGAAACACG
4441GCTTTTCCTACCAAACTCAA
GGAAITATCAAACTCTGCAACACTTGCGTA
TGCAGGTAGC
4501AAGGGAAAIGTCATACTTGA
AGTCGGACAGTGAGTGTAGTCTTGAGAAAT
TCTGAAGCCG
4561
4621
4681
4741
4801
4861
4921
4931
5041
5101
5161
5221
5281
5341
5401
5461
5521
5581
5641
5701
5761
5821
5881
5941
6001
pP1C9K序列
TATTTTTATTA1CAGTGAGT
CAGTCATCAGGAGATCCTCTAOGCCGGACGCATCGTGGCC
GACCTGCAGGGGGGGGGGGG
GCGCTGAGGTCTGCCTCGTGAAGAAGGTGTTGCTGACTCA
TACCAGGCCTGAATCGCCCC
ATCATCCAGCCAGAAAGTGAGGGAGCCACGGTTGATGAGA
GCTTTGTTGTAGGTGGACCA
GTTGGTGATTTTGAACTTTTGCT