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- 详细信息
- 询价记录
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pPIC9K
- 库存：
100
- 供应商：
上海烜雅生物科技有限公司
- 规格：
干粉/液体
基本信息
名称：pPIC9k毕赤酵母表达质粒
别称: pPIC9K
质粒属性
质粒简介
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。1.载体大小9276 bp。2.载体构建过程中，可供使用的单一限制性内切酶位点为 SnaB I, EcoR I, Avr II, Not I. 3.利用alpha因子分泌信号肽，分泌表达蛋白基因。4.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。5.毕赤酵母中利用HIS4进行筛选。6.为了将基因插入GS115或KM71的AOX区, 使用SacI限制性内切酶线性化质粒(GS115中产生His+Mut+基因型，KM71中产生His+ MutS基因型) 7..为了将基因插入GS115或KM71的His4区, 使用Sal I或者Stu I限制性内切酶线性化质粒(GS115中产生His+ Mut+基因型，KM71中产生His+ MutS基因型) 8.将基因插入GS115的AOX1区域，需要使用Bgl II线性化质粒 (产生His+MutS基因型)。
质粒图谱
质粒序列
LOCUS Exported 9276 bp ds-DNA circular SYN 10-SEP-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled 2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9276)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..9276
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 2..937
/gene="Pichia pastoris AOX1"
/note="AOX1 promoter"
/note="inducible promoter, regulated by methanol"
CDS 949..1215
/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."
/translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA"
terminator 1321..1567
/gene="Pichia pastoris AOX1"
/note="AOX1 terminator"
/note="transcription terminator for AOX1"
CDS complement(1980..4514)
/codon_start=1
/gene="Pichia pastoris HIS4"
/product="multifunctional enzyme, required for histidine
biosynthesis"
/note="PpHIS4"
/note="auxotrophic marker for Pichia pastoris"
/translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE
LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL
VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII
DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI
PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER
TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED
IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP
KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE
LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVET-CP
GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE
VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA
LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED
AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV
QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK
HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"
CDS complement(4928..5743)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
misc_feature 6122..6878
/note="AOX1 3' fragment"
/note="region downstream of Pichia pastoris AOX1 gene"
misc_feature 7021..7161
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(7347..7935)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8106..8966)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(8967..9071)
/gene="bla"
/note="AmpR promoter"
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 aaagtcgcca 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 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 atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa
1321 tcaagaggat gtcagaatgc catttgcctg 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 agtgagaagt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt ttatcacagt
1621 taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc
1681 tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc
1741 tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc
1801 tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg
1861 gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac gcgatcatgg
1921 cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc tctaaataat
1981 taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc atctagacct
2041 tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc aggagttacg
2101 tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta ttgacgggca
2161 tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca actctctgga
2221 gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata agaagaagca
2281 ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc caaagcctgc
2341 tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac aatttcaacc
2401 cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag ctccttgtct
2461 gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc ttgagacaga
2521 aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag aacttcagaa
2581 ggcccagcag gcatgtcaat actacacagg gctgatgtgt cattttgaac catcatcttg
2641 gcagcagtaa cgaactggtt tcctggacca aatattttgt cacacttagg aacagtttct
2701 gttccgtaag ccatagcagc tactgcctgg gcgcctcctg ctagcacgat acacttagca
2761 ccaaccttgt gggcaacgta gatgacttct ggggtaaggg taccatcctt cttaggtgga
2821 gatgcaaaaa caatttcttt gcaaccagca actttggcag gaacacccag catcagggaa
2881 gtggaaggca gaattgcggt tccaccagga atatagaggc caactttctc aataggtctt
2941 gcaaaacgag agcagactac accagggcaa gtctcaactt gcaacgtctc cgttagttga
3001 gcttcatgga atttcctgac gttatctata gagagatcaa tggctctctt aacgttatct
3061 ggcaattgca taagttcctc tgggaaagga gcttctaaca caggtgtctt caaagcgact
3121 ccatcaaact tggcagttag ttctaaaagg gctttgtcac cattttgacg aacattgtcg
3181 acaattggtt tgactaattc cataatctgt tccgttttct ggataggacg acgaagggca
3241 tcttcaattt cttgtgagga ggccttagaa acgtcaattt tgcacaattc aatacgacct
3301 tcagaaggga cttctttagg tttggattct tctttaggtt gttccttggt gtatcctggc
3361 ttggcatctc ctttccttct agtgaccttt agggacttca tatccaggtt tctctccacc
3421 tcgtccaacg tcacaccgta cttggcacat ctaactaatg caaaataaaa taagtcagca
3481 cattcccagg ctatatcttc cttggattta gcttctgcaa gttcatcagc ttcctcccta
3541 attttagcgt tcaacaaaac ttcgtcgtca aataaccgtt tggtataaga accttctgga
3601 gcattgctct tacgatccca caaggtggct tccatggctc taagaccctt tgattggcca
3661 aaacaggaag tgcgttccaa gtgacagaaa ccaacacctg tttgttcaac cacaaatttc
3721 aagcagtctc catcacaatc caattcgata cccagcaact tttgagttgc tccagatgta
3781 gcacctttat accacaaacc gtgacgacga gattggtaga ctccagtttg tgtccttata
3841 gcctccggaa tagacttttt ggacgagtac accaggccca acgagtaatt agaagagtca
3901 gccaccaaag tagtgaatag accatcgggg cggtcagtag tcaaagacgc caacaaaatt
3961 tcactgacag ggaacttttt gacatcttca gaaagttcgt attcagtagt caattgccga
4021 gcatcaataa tggggattat accagaagca acagtggaag tcacatctac caactttgcg
4081 gtctcagaaa aagcataaac agttctacta ccgccattag tgaaactttt caaatcgccc
4141 agtggagaag aaaaaggcac agcgatacta gcattagcgg gcaaggatgc aactttatca
4201 accagggtcc tatagataac cctagcgcct gggatcatcc tttggacaac tctttctgcc
4261 aaatctaggt ccaaaatcac ttcattgata ccattattgt acaacttgag caagttgtcg
4321 atcagctcct caaattggtc ctctgtaacg gatgactcaa cttgcacatt aacttgaagc
4381 tcagtcgatt gagtgaactt gatcaggttg tgcagctggt cagcagcata gggaaacacg
4441 gcttttccta ccaaactcaa ggaattatca aactctgcaa cacttgcgta tgcaggtagc
4501 aagggaaatg tcatacttga agtcggacag tgagtgtagt cttgagaaat tctgaagccg
4561 tatttttatt atcagtgagt cagtcatcag gagatcctct acgccggacg catcgtggcc
4621 gacctgcagg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca
4681 taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga
4741 gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct
4801 gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa
4861 caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac caattaacca
4921 attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
4981 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
5041 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
5101 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
5161 tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa
5221 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
5281 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
5341 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
5401 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
5461 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
5521 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
5581 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
5641 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
5701 atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
5761 tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt
5821 aacatcagag attttgagac acaacgtggc tttccccccc ccccctgcag gtcggcatca
5881 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
5941 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
6001 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
6061 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
6121 gtcgagtatc tatgattgga agtatgggaa tggtgatacc cgcattcttc agtgtcttga
6181 ggtctcctat cagattatgc ccaactaaag caaccggagg aggagatttc atggtaaatt
6241 tctctgactt ttggtcatca gtagactcga actgtgagac tatctcggtt atgacagcag
6301 aaatgtcctt cttggagaca gtaaatgaag tcccaccaat aaagaaatcc ttgttatcag
6361 gaacaaactt cttgtttcga actttttcgg tgccttgaac tataaaatgt agagtggata
6421 tgtcgggtag gaatggagcg ggcaaatgct taccttctgg accttcaaga ggtatgtagg
6481 gtttgtagat actgatgcca acttcagtga caacgttgct atttcgttca aaccattccg
6541 aatccagaga aatcaaagtt gtttgtctac tattgatcca agccagtgcg gtcttgaaac
6601 tgacaatagt gtgctcgtgt tttgaggtca tctttgtatg aataaatcta gtctttgatc
6661 taaataatct tgacgagcca aggcgataaa tacccaaatc taaaactctt ttaaaacgtt
6721 aaaaggacaa gtatgtctgc ctgtattaaa ccccaaatca gctcgtagtc tgatcctcat
6781 caacttgagg ggcactatct tgttttagag aaatttgcgg agatgcgata tcgagaaaaa
6841 ggtacgctga ttttaaacgt gaaatttatc tcaagatctc tgcctcgcgc gtttcggtga
6901 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
6961 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
7021 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
7081 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
7141 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
7201 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
7261 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
7321 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
7381 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
7441 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
7501 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat
7561 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
7621 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
7681 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
7741 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
7801 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
7861 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
7921 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
7981 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
8041 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
8101 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
8161 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
8221 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
8281 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
8341 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
8401 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
8461 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
8521 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
8581 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
8641 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
8701 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa
8761 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
8821 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
8881 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
8941 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
9001 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
9061 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
9121 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt
9181 ctcatgtttg acagcttatc atcgataagc tgactcatgt tggtattgtg aaatagacgc
9241 agatcgggaa cactgaaaaa taacagttat tattcg
//
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1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
名称：pPIC9k毕赤酵母表达质粒
别称: pPIC9K
| 启动子: | AOX1 |
|---|---|
| 复制子: | pUC |
| 质粒分类: | 酵母系列，毕赤酵母表达载体 |
| 质粒大小: | 9276bp |
| 原核抗性: | Amp,Kan |
| 筛选标记: | HIS4,G418 |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 酵母细胞 |
| 5'测序引物: | 5´AOX:GACTGGTTCCAATTGACAAGC |
| 3'测序引物: | 3´AOX:GGCAAATGGCATTCTGACAT |
| 备注: | 利用alpha分泌因子，分泌表达蛋白。 |
质粒属性
| 载体宿主： | 酵母菌 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | 空载体 |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 筛选标记： | Neo/G418 |
| 荧光蛋白： |
质粒简介
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。1.载体大小9276 bp。2.载体构建过程中，可供使用的单一限制性内切酶位点为 SnaB I, EcoR I, Avr II, Not I. 3.利用alpha因子分泌信号肽，分泌表达蛋白基因。4.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。5.毕赤酵母中利用HIS4进行筛选。6.为了将基因插入GS115或KM71的AOX区, 使用SacI限制性内切酶线性化质粒(GS115中产生His+Mut+基因型，KM71中产生His+ MutS基因型) 7..为了将基因插入GS115或KM71的His4区, 使用Sal I或者Stu I限制性内切酶线性化质粒(GS115中产生His+ Mut+基因型，KM71中产生His+ MutS基因型) 8.将基因插入GS115的AOX1区域，需要使用Bgl II线性化质粒 (产生His+MutS基因型)。
质粒图谱
质粒序列
LOCUS Exported 9276 bp ds-DNA circular SYN 10-SEP-2016
DEFINITION synthetic circular DNA
KEYWORDS Untitled 2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9276)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..9276
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 2..937
/gene="Pichia pastoris AOX1"
/note="AOX1 promoter"
/note="inducible promoter, regulated by methanol"
CDS 949..1215
/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."
/translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA"
terminator 1321..1567
/gene="Pichia pastoris AOX1"
/note="AOX1 terminator"
/note="transcription terminator for AOX1"
CDS complement(1980..4514)
/codon_start=1
/gene="Pichia pastoris HIS4"
/product="multifunctional enzyme, required for histidine
biosynthesis"
/note="PpHIS4"
/note="auxotrophic marker for Pichia pastoris"
/translation="MTFPLLPAYASVAEFDNSLSLVGKAVFPYAADQLHNLIKFTQSTE
LQVNVQVESSVTEDQFEELIDNLLKLYNNGINEVILDLDLAERVVQRMIPGARVIYRTL
VDKVASLPANASIAVPFSSPLGDLKSFTNGGSRTVYAFSETAKLVDVTSTVASGIIPII
DARQLTTEYELSEDVKKFPVSEILLASLTTDRPDGLFTTLVADSSNYSLGLVYSSKKSI
PEAIRTQTGVYQSRRHGLWYKGATSGATQKLLGIELDCDGDCLKFVVEQTGVGFCHLER
TSCFGQSKGLRAMEATLWDRKSNAPEGSYTKRLFDDEVLLNAKIREEADELAEAKSKED
IAWECADLFYFALVRCAKYGVTLDEVERNLDMKSLKVTRRKGDAKPGYTKEQPKEESKP
KEVPSEGRIELCKIDVSKASSQEIEDALRRPIQKTEQIMELVKPIVDNVRQNGDKALLE
LTAKFDGVALKTPVLEAPFPEELMQLPDNVKRAIDLSIDNVRKFHEAQLTETLQVET-CP
GVVCSRFARPIEKVGLYIPGGTAILPSTSLMLGVPAKVAGCKEIVFASPPKKDGTLTPE
VIYVAHKVGAKCIVLAGGAQAVAAMAYGTETVPKCDKIFGPGNQFVTAAKMMVQNDTSA
LCSIDMPAGPSEVLVIADKYADPDFVASDLLSQAEHGIDSQVILLAVDMTDKELARIED
AVHNQAVQLPRVEIVRKCIAHSTTLSVATYEQALEMSNQYAPEHLILQIENASSYVDQV
QHAGSVFVGAYSPESCGDYSSGTNHTLPTYGYARQYSGVNTATFQKFITSQDVTPEGLK
HIGQAVMDLAAVEGLDAHRNAVKVRMEKLGLI"
CDS complement(4928..5743)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/note="KanR"
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
misc_feature 6122..6878
/note="AOX1 3' fragment"
/note="region downstream of Pichia pastoris AOX1 gene"
misc_feature 7021..7161
/note="bom"
/note="basis of mobility region from pBR322"
rep_origin complement(7347..7935)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8106..8966)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(8967..9071)
/gene="bla"
/note="AmpR promoter"
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 aaagtcgcca 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 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 atgactgttc ctcagttcaa gttgggcact tacgagaaga ccggtcttgc tagattctaa
1321 tcaagaggat gtcagaatgc catttgcctg 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 agtgagaagt tcgtttgtgc aagcttatcg ataagcttta atgcggtagt ttatcacagt
1621 taaattgcta acgcagtcag gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc
1681 tcggcaccgt caccctggat gctgtaggca taggcttggt tatgccggta ctgccgggcc
1741 tcttgcggga tatcgtccat tccgacagca tcgccagtca ctatggcgtg ctgctagcgc
1801 tatatgcgtt gatgcaattt ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg
1861 gccgccgccc agtcctgctc gcttcgctac ttggagccac tatcgactac gcgatcatgg
1921 cgaccacacc cgtcctgtgg atctatcgaa tctaaatgta agttaaaatc tctaaataat
1981 taaataagtc ccagtttctc catacgaacc ttaacagcat tgcggtgagc atctagacct
2041 tcaacagcag ccagatccat cactgcttgg ccaatatgtt tcagtccctc aggagttacg
2101 tcttgtgaag tgatgaactt ctggaaggtt gcagtgttaa ctccgctgta ttgacgggca
2161 tatccgtacg ttggcaaagt gtggttggta ccggaggagt aatctccaca actctctgga
2221 gagtaggcac caacaaacac agatccagcg tgttgtactt gatcaacata agaagaagca
2281 ttctcgattt gcaggatcaa gtgttcagga gcgtactgat tggacatttc caaagcctgc
2341 tcgtaggttg caaccgatag ggttgtagag tgtgcaatac acttgcgtac aatttcaacc
2401 cttggcaact gcacagcttg gttgtgaaca gcatcttcaa ttctggcaag ctccttgtct
2461 gtcatatcga cagccaacag aatcacctgg gaatcaatac catgttcagc ttgagacaga
2521 aggtctgagg caacgaaatc tggatcagcg tatttatcag caataactag aacttcagaa
2581 ggcccagcag gcatgtcaat actacacagg gctgatgtgt cattttgaac catcatcttg
2641 gcagcagtaa cgaactggtt tcctggacca aatattttgt cacacttagg aacagtttct
2701 gttccgtaag ccatagcagc tactgcctgg gcgcctcctg ctagcacgat acacttagca
2761 ccaaccttgt gggcaacgta gatgacttct ggggtaaggg taccatcctt cttaggtgga
2821 gatgcaaaaa caatttcttt gcaaccagca actttggcag gaacacccag catcagggaa
2881 gtggaaggca gaattgcggt tccaccagga atatagaggc caactttctc aataggtctt
2941 gcaaaacgag agcagactac accagggcaa gtctcaactt gcaacgtctc cgttagttga
3001 gcttcatgga atttcctgac gttatctata gagagatcaa tggctctctt aacgttatct
3061 ggcaattgca taagttcctc tgggaaagga gcttctaaca caggtgtctt caaagcgact
3121 ccatcaaact tggcagttag ttctaaaagg gctttgtcac cattttgacg aacattgtcg
3181 acaattggtt tgactaattc cataatctgt tccgttttct ggataggacg acgaagggca
3241 tcttcaattt cttgtgagga ggccttagaa acgtcaattt tgcacaattc aatacgacct
3301 tcagaaggga cttctttagg tttggattct tctttaggtt gttccttggt gtatcctggc
3361 ttggcatctc ctttccttct agtgaccttt agggacttca tatccaggtt tctctccacc
3421 tcgtccaacg tcacaccgta cttggcacat ctaactaatg caaaataaaa taagtcagca
3481 cattcccagg ctatatcttc cttggattta gcttctgcaa gttcatcagc ttcctcccta
3541 attttagcgt tcaacaaaac ttcgtcgtca aataaccgtt tggtataaga accttctgga
3601 gcattgctct tacgatccca caaggtggct tccatggctc taagaccctt tgattggcca
3661 aaacaggaag tgcgttccaa gtgacagaaa ccaacacctg tttgttcaac cacaaatttc
3721 aagcagtctc catcacaatc caattcgata cccagcaact tttgagttgc tccagatgta
3781 gcacctttat accacaaacc gtgacgacga gattggtaga ctccagtttg tgtccttata
3841 gcctccggaa tagacttttt ggacgagtac accaggccca acgagtaatt agaagagtca
3901 gccaccaaag tagtgaatag accatcgggg cggtcagtag tcaaagacgc caacaaaatt
3961 tcactgacag ggaacttttt gacatcttca gaaagttcgt attcagtagt caattgccga
4021 gcatcaataa tggggattat accagaagca acagtggaag tcacatctac caactttgcg
4081 gtctcagaaa aagcataaac agttctacta ccgccattag tgaaactttt caaatcgccc
4141 agtggagaag aaaaaggcac agcgatacta gcattagcgg gcaaggatgc aactttatca
4201 accagggtcc tatagataac cctagcgcct gggatcatcc tttggacaac tctttctgcc
4261 aaatctaggt ccaaaatcac ttcattgata ccattattgt acaacttgag caagttgtcg
4321 atcagctcct caaattggtc ctctgtaacg gatgactcaa cttgcacatt aacttgaagc
4381 tcagtcgatt gagtgaactt gatcaggttg tgcagctggt cagcagcata gggaaacacg
4441 gcttttccta ccaaactcaa ggaattatca aactctgcaa cacttgcgta tgcaggtagc
4501 aagggaaatg tcatacttga agtcggacag tgagtgtagt cttgagaaat tctgaagccg
4561 tatttttatt atcagtgagt cagtcatcag gagatcctct acgccggacg catcgtggcc
4621 gacctgcagg gggggggggg gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca
4681 taccaggcct gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga
4741 gctttgttgt aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct
4801 gcgttgtcgg gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa
4861 caaagccgcc gtcccgtcaa gtcagcgtaa tgctctgcca gtgttacaac caattaacca
4921 attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
4981 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
5041 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
5101 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
5161 tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa
5221 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
5281 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
5341 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
5401 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
5461 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
5521 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
5581 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
5641 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
5701 atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
5761 tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt
5821 aacatcagag attttgagac acaacgtggc tttccccccc ccccctgcag gtcggcatca
5881 ccggcgccac aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc
5941 gggctcgcca cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg
6001 tggccggggg actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc
6061 tcaacggcct caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc
6121 gtcgagtatc tatgattgga agtatgggaa tggtgatacc cgcattcttc agtgtcttga
6181 ggtctcctat cagattatgc ccaactaaag caaccggagg aggagatttc atggtaaatt
6241 tctctgactt ttggtcatca gtagactcga actgtgagac tatctcggtt atgacagcag
6301 aaatgtcctt cttggagaca gtaaatgaag tcccaccaat aaagaaatcc ttgttatcag
6361 gaacaaactt cttgtttcga actttttcgg tgccttgaac tataaaatgt agagtggata
6421 tgtcgggtag gaatggagcg ggcaaatgct taccttctgg accttcaaga ggtatgtagg
6481 gtttgtagat actgatgcca acttcagtga caacgttgct atttcgttca aaccattccg
6541 aatccagaga aatcaaagtt gtttgtctac tattgatcca agccagtgcg gtcttgaaac
6601 tgacaatagt gtgctcgtgt tttgaggtca tctttgtatg aataaatcta gtctttgatc
6661 taaataatct tgacgagcca aggcgataaa tacccaaatc taaaactctt ttaaaacgtt
6721 aaaaggacaa gtatgtctgc ctgtattaaa ccccaaatca gctcgtagtc tgatcctcat
6781 caacttgagg ggcactatct tgttttagag aaatttgcgg agatgcgata tcgagaaaaa
6841 ggtacgctga ttttaaacgt gaaatttatc tcaagatctc tgcctcgcgc gtttcggtga
6901 tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc
6961 ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg
7021 cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca
7081 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
7141 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
7201 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
7261 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
7321 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
7381 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
7441 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
7501 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat
7561 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
7621 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
7681 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
7741 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
7801 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
7861 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
7921 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
7981 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
8041 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
8101 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
8161 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
8221 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
8281 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
8341 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
8401 cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct
8461 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
8521 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
8581 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
8641 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
8701 agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa
8761 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
8821 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
8881 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
8941 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
9001 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
9061 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
9121 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaattaatt
9181 ctcatgtttg acagcttatc atcgataagc tgactcatgt tggtattgtg aaatagacgc
9241 agatcgggaa cactgaaaaa taacagttat tattcg
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
| P3020/xCas9 3.7 哺乳编辑质粒 |
| P3021/xCas9(3.7)-BE3哺乳编辑质粒 |
| P3475/pC015 - dLwCas13a-NF哺乳编辑质粒 |
| P3022/pC0056 - LwCas13a-msfGFP-NES哺乳编辑质粒 |
| P4950/pXR002-EF1a-dCasRx-2A-EGFP哺乳编辑质粒 |
| P4947/pXR004-CasRx pre-gRNA cloning backbone哺乳编辑质粒 |
| P3203/pY30哺乳编辑质粒 |
| P0665/NLS-NgAgo-pCDNA3.1哺乳编辑质粒 |
| P0666/pCDNA3.1-NLS-NgAgo哺乳编辑质粒 |
| P0687/NLS-NgAgo-GK哺乳编辑质粒 |
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pPIC9k毕赤酵母表达质粒
￥100 - 1000
