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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
NM_001430.4;EPAS1;bHLHe73;ECYT4;HIF2
- 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：pCDH-CMV-MCS-EF1-Puro-FLAG-HIF2α人源基因质粒
别称: NM_001430.4;EPAS1;bHLHe73;ECYT4;HIF2
启动子：CMV
复制子: pUC
原核抗性: Amp
筛选标记：Puro
克隆菌株: Stbl3
培养条件: 37度
质粒属性
载体宿主：哺乳细胞
载体用途：蛋白表达
基因种属：人
基因类型：ORF
原核抗性：Amp
真核抗性：Puro
荧光蛋白：
质粒简介
pCDH-CMV-MCS-EF1-Puro-Flag-HIF2α人源基因质粒
质粒图谱
质粒序列
LOCUS Exported 10010 bp ds-DNA circular SYN 06-JUL-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pCDH-CMV-MCS-EF1-Puro-Flag-HIF2-alpha
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10010)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..10010
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 234..414
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 458..583
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1076..1309
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 1804..1920
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2010..2213
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
regulatory 2290..2299
/regulatory_class="other"
/note="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 2299..2322
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/note="FLAG"
/translation="DYKDDDDK"
misc_feature 2323..4932
/note="HIF2-alpha"
promoter 4980..5191
/note="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
LTR 5204..5472
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
CDS 5499..6098
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 6105..6693
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 6767..7000
/note="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 7072..7193
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 7212..7347
/note="SV40 ori"
/note="SV40 origin of replication"
rep_origin complement(7787..8375)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8546..9406)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(9407..9511)
/gene="bla"
/note="AmpR promoter"
ORIGIN
1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc
241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
421 cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct
481 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
541 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
601 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
661 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
721 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
781 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
841 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
901 taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg
961 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
1021 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
1081 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc
1141 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
1201 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
1261 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
1321 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
1381 aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa
1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt
1801 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
1861 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt
1921 atcgatacta gtattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
1981 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
2041 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
2101 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
2161 tgacgcaaat gggcggtagg cgtgtacggt gggaggttta tataagcaga gctcgtttag
2221 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagattct
2281 agagctagcg ccaccatgga ttacaaggat gacgacgata agacagctga caaggagaag
2341 aaaaggagta gctcggagag gaggaaggag aagtcccggg atgctgcgcg gtgccggcgg
2401 agcaaggaga cggaggtgtt ctatgagctg gcccatgagc tgcctctgcc ccacagtgtg
2461 agctcccatc tggacaaggc ctccatcatg cgactggcaa tcagcttcct gcgaacacac
2521 aagctcctct cctcagtttg ctctgaaaac gagtccgaag ccgaagctga ccagcagatg
2581 gacaacttgt acctgaaagc cttggagggt ttcattgccg tggtgaccca agatggcgac
2641 atgatctttc tgtcagaaaa catcagcaag ttcatgggac ttacacaggt ggagctaaca
2701 ggacatagta tctttgactt cactcatccc tgcgaccatg aggagattcg tgagaacctg
2761 agtctcaaaa atggctctgg ttttgggaaa aaaagcaaag acatgtccac agagcgggac
2821 ttcttcatga ggatgaagtg cacggtcacc aacagaggcc gtactgtcaa cctcaagtca
2881 gccacctgga aggtcttgca ctgcacgggc caggtgaaag tctacaacaa ctgccctcct
2941 cacaatagtc tgtgtggcta caaggagccc ctgctgtcct gcctcatcat catgtgtgaa
3001 ccaatccagc acccatccca catggacatc cccctggata gcaagacctt cctgagccgc
3061 cacagcatgg acatgaagtt cacctactgt gatgacagaa tcacagaact gattggttac
3121 caccctgagg agctgcttgg ccgctcagcc tatgaattct accatgcgct agactccgag
3181 aacatgacca agagtcacca gaacttgtgc accaagggtc aggtagtaag tggccagtac
3241 cggatgctcg caaagcatgg gggctacgtg tggctggaga cccaggggac ggtcatctac
3301 aaccctcgca acctgcagcc ccagtgcatc atgtgtgtca actacgtcct gagtgagatt
3361 gagaagaatg acgtggtgtt ctccatggac cagactgaat ccctgttcaa gccccacctg
3421 atggccatga acagcatctt tgatagcagt ggcaaggggg ctgtgtctga gaagagtaac
3481 ttcctattca ccaagctaaa ggaggagccc gaggagctgg cccagctggc tcccacccca
3541 ggagacgcca tcatctctct ggatttcggg aatcagaact tcgaggagtc ctcagcctat
3601 ggcaaggcca tcctgccccc gagccagcca tgggccacgg agttgaggag ccacagcacc
3661 cagagcgagg ctgggagcct gcctgccttc accgtgcccc aggcagctgc cccgggcagc
3721 accaccccca gtgccaccag cagcagcagc agctgctcca cgcccaatag ccctgaagac
3781 tattacacat ctttggataa cgacctgaag attgaagtga ttgagaagct cttcgccatg
3841 gacacagagg ccaaggacca atgcagtacc cagacggatt tcaatgagct ggacttggag
3901 acactggcac cctatatccc catggacggg gaagacttcc agctaagccc catctgcccc
3961 gaggagcggc tcttggcgga gaacccacag tccacccccc agcactgctt cagtgccatg
4021 acaaacatct tccagccact ggcccctgta gccccgcaca gtcccttcct cctggacaag
4081 tttcagcagc agctggagag caagaagaca gagcccgagc accggcccat gtcctccatc
4141 ttctttgatg ccggaagcaa agcatccctg ccaccgtgct gtggccaggc cagcacccct
4201 ctctcttcca tggggggcag atccaatacc cagtggcccc cagatccacc attacatttt
4261 gggcccacaa agtgggccgt cggggatcag cgcacagagt tcttgggagc agcgccgttg
4321 gggccccctg tctctccacc ccatgtctcc accttcaaga caaggtctgc aaagggtttt
4381 ggggctcgag gcccagacgt gctgagtccg gccatggtag ccctctccaa caagctgaag
4441 ctgaagcgac agctggagta tgaagagcaa gccttccagg acctgagcgg gggggaccca
4501 cctggtggca gcacctcaca tttgatgtgg aaacggatga agaacctcag gggtgggagc
4561 tgccctttga tgccggacaa gccactgagc gcaaatgtac ccaatgataa gttcacccaa
4621 aaccccatga ggggcctggg ccatcccctg agacatctgc cgctgccaca gcctccatct
4681 gccatcagtc ccggggagaa cagcaagagc aggttccccc cacagtgcta cgccacccag
4741 taccaggact acagcctgtc gtcagcccac aaggtgtcag gcatggcaag ccggctgctc
4801 gggccctcat ttgagtccta cctgctgccc gaactgacca gatatgactg tgaggtgaac
4861 gtgcccgtgc tgggaagctc cacgctcctg caaggagggg acctcctcag agccctggac
4921 caggccacct gaggatccgc ggccgcgaag gatctgcgat cgctccggtg cccgtcagtg
4981 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
5041 gggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
5101 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
5161 ttttcgcaac gggtttgccg ccagaacaca gctgaagctt cgaggggctc gcatctctcc
5221 ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc cggttgagtc gcgttctgcc
5281 gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc taggtaagtt taaagctcag
5341 gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca gccggctctc
5401 cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt ctgttctgcg
5461 ccgttacaga tccaagctgt gaccggcgcc tacgctagat gaccgagtac aagcccacgg
5521 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
5581 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
5641 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
5701 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
5761 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
5821 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
5881 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
5941 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
6001 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
6061 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgagt cgacaatcaa cctctggatt
6121 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
6181 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
6241 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
6301 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
6361 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
6421 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
6481 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
6541 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
6601 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
6661 cgagtcggat ctccctttgg gccgcctccc cgcctggtac ctttaagacc aatgacttac
6721 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
6781 cactcccaac gaaaataaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
6841 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
6901 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
6961 tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt
7021 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
7081 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
7141 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
7201 tggctctagc tatcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
7261 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
7321 agtagtgagg aggctttttt ggaggcctag acttttgcag agacggccca aattcgtaat
7381 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
7441 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
7501 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
7561 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
7621 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
7681 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
7741 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
7801 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
7861 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
7921 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
7981 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
8041 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
8101 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
8161 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
8221 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
8281 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
8341 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
8401 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
8461 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
8521 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
8581 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
8641 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
8701 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
8761 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
8821 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
8881 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
8941 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
9001 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
9061 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
9121 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
9181 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
9241 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
9301 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
9361 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
9421 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
9481 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
9541 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
9601 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
9661 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
9721 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
9781 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
9841 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
9901 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
9961 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgccaagctg
//
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
名称：pCDH-CMV-MCS-EF1-Puro-FLAG-HIF2α人源基因质粒
别称: NM_001430.4;EPAS1;bHLHe73;ECYT4;HIF2
启动子：CMV
复制子: pUC
原核抗性: Amp
筛选标记：Puro
克隆菌株: Stbl3
培养条件: 37度
质粒属性
载体宿主：哺乳细胞
载体用途：蛋白表达
基因种属：人
基因类型：ORF
原核抗性：Amp
真核抗性：Puro
荧光蛋白：
质粒简介
pCDH-CMV-MCS-EF1-Puro-Flag-HIF2α人源基因质粒
质粒图谱
质粒序列
LOCUS Exported 10010 bp ds-DNA circular SYN 06-JUL-2018
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS pCDH-CMV-MCS-EF1-Puro-Flag-HIF2-alpha
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 10010)
AUTHORS .
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..10010
/organism="synthetic DNA construct"
/mol_type="other DNA"
LTR 234..414
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 458..583
/note="HIV-1 Psi"
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1076..1309
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 1804..1920
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2010..2213
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
regulatory 2290..2299
/regulatory_class="other"
/note="Kozak sequence"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 2299..2322
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/note="FLAG"
/translation="DYKDDDDK"
misc_feature 2323..4932
/note="HIF2-alpha"
promoter 4980..5191
/note="EF-1-alpha core promoter"
/note="core promoter for human elongation factor
EF-1-alpha"
LTR 5204..5472
/note="5' LTR (truncated)"
/note="truncated 5' long terminal repeat (LTR) from human
T-cell leukemia virus (HTLV) type 1"
CDS 5499..6098
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/note="PuroR"
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 6105..6693
/note="WPRE"
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 6767..7000
/note="3' LTR (Delta-U3)"
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 7072..7193
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin 7212..7347
/note="SV40 ori"
/note="SV40 origin of replication"
rep_origin complement(7787..8375)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8546..9406)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(9407..9511)
/gene="bla"
/note="AmpR promoter"
ORIGIN
1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc
241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
421 cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct
481 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
541 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
601 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
661 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
721 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
781 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
841 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
901 taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg
961 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
1021 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
1081 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc
1141 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
1201 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
1261 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
1321 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
1381 aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa
1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt
1801 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
1861 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt
1921 atcgatacta gtattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
1981 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
2041 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
2101 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
2161 tgacgcaaat gggcggtagg cgtgtacggt gggaggttta tataagcaga gctcgtttag
2221 tgaaccgtca gatcgcctgg agacgccatc cacgctgttt tgacctccat agaagattct
2281 agagctagcg ccaccatgga ttacaaggat gacgacgata agacagctga caaggagaag
2341 aaaaggagta gctcggagag gaggaaggag aagtcccggg atgctgcgcg gtgccggcgg
2401 agcaaggaga cggaggtgtt ctatgagctg gcccatgagc tgcctctgcc ccacagtgtg
2461 agctcccatc tggacaaggc ctccatcatg cgactggcaa tcagcttcct gcgaacacac
2521 aagctcctct cctcagtttg ctctgaaaac gagtccgaag ccgaagctga ccagcagatg
2581 gacaacttgt acctgaaagc cttggagggt ttcattgccg tggtgaccca agatggcgac
2641 atgatctttc tgtcagaaaa catcagcaag ttcatgggac ttacacaggt ggagctaaca
2701 ggacatagta tctttgactt cactcatccc tgcgaccatg aggagattcg tgagaacctg
2761 agtctcaaaa atggctctgg ttttgggaaa aaaagcaaag acatgtccac agagcgggac
2821 ttcttcatga ggatgaagtg cacggtcacc aacagaggcc gtactgtcaa cctcaagtca
2881 gccacctgga aggtcttgca ctgcacgggc caggtgaaag tctacaacaa ctgccctcct
2941 cacaatagtc tgtgtggcta caaggagccc ctgctgtcct gcctcatcat catgtgtgaa
3001 ccaatccagc acccatccca catggacatc cccctggata gcaagacctt cctgagccgc
3061 cacagcatgg acatgaagtt cacctactgt gatgacagaa tcacagaact gattggttac
3121 caccctgagg agctgcttgg ccgctcagcc tatgaattct accatgcgct agactccgag
3181 aacatgacca agagtcacca gaacttgtgc accaagggtc aggtagtaag tggccagtac
3241 cggatgctcg caaagcatgg gggctacgtg tggctggaga cccaggggac ggtcatctac
3301 aaccctcgca acctgcagcc ccagtgcatc atgtgtgtca actacgtcct gagtgagatt
3361 gagaagaatg acgtggtgtt ctccatggac cagactgaat ccctgttcaa gccccacctg
3421 atggccatga acagcatctt tgatagcagt ggcaaggggg ctgtgtctga gaagagtaac
3481 ttcctattca ccaagctaaa ggaggagccc gaggagctgg cccagctggc tcccacccca
3541 ggagacgcca tcatctctct ggatttcggg aatcagaact tcgaggagtc ctcagcctat
3601 ggcaaggcca tcctgccccc gagccagcca tgggccacgg agttgaggag ccacagcacc
3661 cagagcgagg ctgggagcct gcctgccttc accgtgcccc aggcagctgc cccgggcagc
3721 accaccccca gtgccaccag cagcagcagc agctgctcca cgcccaatag ccctgaagac
3781 tattacacat ctttggataa cgacctgaag attgaagtga ttgagaagct cttcgccatg
3841 gacacagagg ccaaggacca atgcagtacc cagacggatt tcaatgagct ggacttggag
3901 acactggcac cctatatccc catggacggg gaagacttcc agctaagccc catctgcccc
3961 gaggagcggc tcttggcgga gaacccacag tccacccccc agcactgctt cagtgccatg
4021 acaaacatct tccagccact ggcccctgta gccccgcaca gtcccttcct cctggacaag
4081 tttcagcagc agctggagag caagaagaca gagcccgagc accggcccat gtcctccatc
4141 ttctttgatg ccggaagcaa agcatccctg ccaccgtgct gtggccaggc cagcacccct
4201 ctctcttcca tggggggcag atccaatacc cagtggcccc cagatccacc attacatttt
4261 gggcccacaa agtgggccgt cggggatcag cgcacagagt tcttgggagc agcgccgttg
4321 gggccccctg tctctccacc ccatgtctcc accttcaaga caaggtctgc aaagggtttt
4381 ggggctcgag gcccagacgt gctgagtccg gccatggtag ccctctccaa caagctgaag
4441 ctgaagcgac agctggagta tgaagagcaa gccttccagg acctgagcgg gggggaccca
4501 cctggtggca gcacctcaca tttgatgtgg aaacggatga agaacctcag gggtgggagc
4561 tgccctttga tgccggacaa gccactgagc gcaaatgtac ccaatgataa gttcacccaa
4621 aaccccatga ggggcctggg ccatcccctg agacatctgc cgctgccaca gcctccatct
4681 gccatcagtc ccggggagaa cagcaagagc aggttccccc cacagtgcta cgccacccag
4741 taccaggact acagcctgtc gtcagcccac aaggtgtcag gcatggcaag ccggctgctc
4801 gggccctcat ttgagtccta cctgctgccc gaactgacca gatatgactg tgaggtgaac
4861 gtgcccgtgc tgggaagctc cacgctcctg caaggagggg acctcctcag agccctggac
4921 caggccacct gaggatccgc ggccgcgaag gatctgcgat cgctccggtg cccgtcagtg
4981 ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac
5041 gggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg
5101 cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct
5161 ttttcgcaac gggtttgccg ccagaacaca gctgaagctt cgaggggctc gcatctctcc
5221 ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc cggttgagtc gcgttctgcc
5281 gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc taggtaagtt taaagctcag
5341 gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca gccggctctc
5401 cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt ctgttctgcg
5461 ccgttacaga tccaagctgt gaccggcgcc tacgctagat gaccgagtac aagcccacgg
5521 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
5581 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
5641 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
5701 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
5761 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
5821 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
5881 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
5941 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
6001 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
6061 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgagt cgacaatcaa cctctggatt
6121 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg
6181 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct
6241 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc
6301 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca
6361 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac
6421 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt
6481 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct
6541 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc
6601 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga
6661 cgagtcggat ctccctttgg gccgcctccc cgcctggtac ctttaagacc aatgacttac
6721 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
6781 cactcccaac gaaaataaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
6841 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
6901 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
6961 tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc atgtcatctt
7021 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
7081 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
7141 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
7201 tggctctagc tatcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
7261 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
7321 agtagtgagg aggctttttt ggaggcctag acttttgcag agacggccca aattcgtaat
7381 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
7441 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
7501 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
7561 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
7621 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
7681 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
7741 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
7801 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
7861 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
7921 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
7981 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
8041 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
8101 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
8161 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
8221 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
8281 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
8341 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
8401 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
8461 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
8521 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
8581 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
8641 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
8701 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
8761 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
8821 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
8881 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
8941 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
9001 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
9061 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
9121 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
9181 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
9241 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
9301 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
9361 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
9421 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
9481 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
9541 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
9601 ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga
9661 cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag
9721 cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga
9781 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
9841 ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
9901 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
9961 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgccaagctg
//
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
| P0970/pCMV6-ETV5-S248A-m小鼠基因质粒 |
| P0971/pCMV6-ETV5-K263A-m小鼠基因质粒 |
| P0956/pCMV6-ETV5-K293A-m小鼠基因质粒 |
| P1014/pCMV6-ETV5-S367A-m小鼠基因质粒 |
| P0953/pLV-CMV-wnt3a-m小鼠基因慢病毒质粒 |
| P0954/pRC/PSV-m CBP-HA小鼠基因质粒 |
| P1033/pCDNA3.1-GPR35-m小鼠基因质粒 |
| P2234/pCDNA3.1-Zeo-GPR35-m小鼠基因质粒 |
| P1089/pCDNA6-V5-His-CaV1.2-m小鼠基因质粒 |
| P1126/pECMV-Socs3-m小鼠基因质粒 |
| P2150/TetO-FUW-OSKM小鼠基因质粒 |
| P1273/pLVML-ECMV-Socs3-m小鼠基因慢病毒质粒 |
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￥100 - 1000
