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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：
Plasmid#17477
- 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：pLenti CMV Puro LUC (w168-1) 慢病毒表达质粒
别称: Plasmid#17477
质粒属性
质粒简介
pLenti CMV Puro LUC (w168-1)质粒由上海烜雅生物科技有限公司提供
质粒图谱
质粒序列
LOCUS Exported 9626 bp ds-DNA circular SYN 26-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9626)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 26, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..9626
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 27..131
/gene="bla"
/label=AmpR promoter
CDS 132..992
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1163..1751
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
LTR 2448..2628
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 2672..2797
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 3290..3523
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 4012..4129
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
enhancer 4152..4455
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 4456..4659
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 4764..4788
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 4794..6446
/codon_start=1
/gene="luc+"
/product="firefly luciferase"
/label=luciferase
/note="enhanced luc+ version of the luciferase gene"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
protein_bind complement(6453..6477)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
misc_feature 6508..7096
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
promoter 7110..7609
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 7630..8229
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
LTR 8367..8600
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 8672..8793
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 8812..8947
/label=SV40 ori
/note="SV40 origin of replication"
rep_origin 9169..9624
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
ORIGIN
1 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
61 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
661 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
1021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
1081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
1141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
1201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
1261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
1321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
1381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
1441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
1501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
1561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
1621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
1681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
1741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
1801 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
1861 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
1921 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
1981 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
2041 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
2101 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
2161 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat
2221 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc
2281 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg
2341 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc
2401 gcattgcaga gatattgtat ttaagtgcct agctcgatac aataaacggg tctctctggt
2461 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
2521 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
2581 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
2641 cagggacctg aaagcgaaag ggaaaccaga gctctctcga cgcaggactc ggcttgctga
2701 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
2761 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
2821 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
2881 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
2941 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
3001 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
3061 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
3121 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
3181 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
3241 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
3301 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcctcaatg acgctgacgg
3361 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
3421 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
3481 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
3541 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
3601 tggaacagat tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
3661 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
3721 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
3781 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
3841 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
3901 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
3961 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggttaa cttttaaaag
4021 aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga
4081 catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaatttta tcgataagct
4141 tgggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
4201 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
4261 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
4321 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
4381 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
4441 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
4501 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
4561 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
4621 ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc
4681 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccgact ctagtccagt
4741 gtggtggaat tctgcagata tcaacaagtt tgtacaaaaa agcaggctcc accatggaag
4801 acgccaaaaa cataaagaaa ggcccggcgc cattctatcc gctggaagat ggaaccgctg
4861 gagagcaact gcataaggct atgaagagat acgccctggt tcctggaaca attgctttta
4921 cagatgcaca tatcgaggtg gacatcactt acgctgagta cttcgaaatg tccgttcggt
4981 tggcagaagc tatgaaacga tatgggctga atacaaatca cagaatcgtc gtatgcagtg
5041 aaaactctct tcaattcttt atgccggtgt tgggcgcgtt atttatcgga gttgcagttg
5101 cgcccgcgaa cgacatttat aatgaacgtg aattgctcaa cagtatgggc atttcgcagc
5161 ctaccgtggt gttcgtttcc aaaaaggggt tgcaaaaaat tttgaacgtg caaaaaaagc
5221 tcccaatcat ccaaaaaatt attatcatgg attctaaaac ggattaccag ggatttcagt
5281 cgatgtacac gttcgtcaca tctcatctac ctcccggttt taatgaatac gattttgtgc
5341 cagagtcctt cgatagggac aagacaattg cactgatcat gaactcctct ggatctactg
5401 gtctgcctaa aggtgtcgct ctgcctcata gaactgcctg cgtgagattc tcgcatgcca
5461 gagatcctat ttttggcaat caaatcattc cggatactgc gattttaagt gttgttccat
5521 tccatcacgg ttttggaatg tttactacac tcggatattt gatatgtgga tttcgagtcg
5581 tcttaatgta tagatttgaa gaagagctgt ttctgaggag ccttcaggat tacaagattc
5641 aaagtgcgct gctggtgcca accctattct ccttcttcgc caaaagcact ctgattgaca
5701 aatacgattt atctaattta cacgaaattg cttctggtgg cgctcccctc tctaaggaag
5761 tcggggaagc ggttgccaag aggttccatc tgccaggtat caggcaagga tatgggctca
5821 ctgagactac atcagctatt ctgattacac ccgaggggga tgataaaccg ggcgcggtcg
5881 gtaaagttgt tccatttttt gaagcgaagg ttgtggatct ggataccggg aaaacgctgg
5941 gcgttaatca aagaggcgaa ctgtgtgtga gaggtcctat gattatgtcc ggttatgtaa
6001 acaatccgga agcgaccaac gccttgattg acaaggatgg atggctacat tctggagaca
6061 tagcttactg ggacgaagac gaacacttct tcatcgttga ccgcctgaag tctctgatta
6121 agtacaaagg ctatcaggtg gctcccgctg aattggaatc catcttgctc caacacccca
6181 acatcttcga cgcaggtgtc gcaggtcttc ccgacgatga cgccggtgaa cttcccgccg
6241 ccgttgttgt tttggagcac ggaaagacga tgacggaaaa agagatcgtg gattacgtcg
6301 ccagtcaagt aacaaccgcg aaaaagttgc gcggaggagt tgtgtttgtg gacgaagtac
6361 cgaaaggtct taccggaaaa ctcgacgcaa gaaaaatcag agagatcctc ataaaggcca
6421 agaagggcgg aaagatcgcc gtgtaattct agacccagct ttcttgtaca aagtggttga
6481 tatccagcac agtggcggcc gctcgacaat caacctctgg attacaaaat ttgtgaaaga
6541 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
6601 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
6661 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
6721 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
6781 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
6841 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
6901 aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
6961 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
7021 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
7081 tgggccgcct ccccgcctgg aattctaccg ggtaggggag gcgcttttcc caaggcagtc
7141 tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg gcctctggcc
7201 tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg gtggcccctt
7261 cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc agctcgcgtc
7321 gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga tggacagcac
7381 cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc tttgctcctt
7441 cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca ggggcgggct
7501 caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa
7561 gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcga cctgcagccc
7621 aagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
7681 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
7741 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
7801 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
7861 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
7921 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
7981 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
8041 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
8101 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
8161 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
8221 ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc acgaccccat
8281 gcatctcgag ggcccggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
8341 ccacttttta aaagaaaagg ggggactgga agggctagct cactcccaac gaagacaaga
8401 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
8461 tggctgccta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
8521 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
8581 agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt atttataact
8641 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
8701 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
8761 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
8821 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
8881 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
8941 ggaggcctag gcttttgcgt cgagacgtac ccaattcgcc ctatagtgag tcgtattacg
9001 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
9061 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
9121 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcgac gcgccctgta
9181 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
9241 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
9301 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
9361 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
9421 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
9481 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
9541 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
9601 acaaaatatt aacgtttaca atttcc
//
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：pLenti CMV Puro LUC (w168-1) 慢病毒表达质粒
别称: Plasmid#17477
| 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,慢病毒 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 筛选标记： | Puro |
| 荧光蛋白： |
pLenti CMV Puro LUC (w168-1)质粒由上海烜雅生物科技有限公司提供
质粒图谱
质粒序列
LOCUS Exported 9626 bp ds-DNA circular SYN 26-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9626)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 26, 2017 from MiaoLingPlasmid
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..9626
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 27..131
/gene="bla"
/label=AmpR promoter
CDS 132..992
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
rep_origin 1163..1751
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
LTR 2448..2628
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 2672..2797
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 3290..3523
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
misc_feature 4012..4129
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
enhancer 4152..4455
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 4456..4659
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
protein_bind 4764..4788
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 4794..6446
/codon_start=1
/gene="luc+"
/product="firefly luciferase"
/label=luciferase
/note="enhanced luc+ version of the luciferase gene"
/translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
protein_bind complement(6453..6477)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
misc_feature 6508..7096
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
promoter 7110..7609
/label=PGK promoter
/note="mouse phosphoglycerate kinase 1 promoter"
CDS 7630..8229
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
LTR 8367..8600
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 8672..8793
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin 8812..8947
/label=SV40 ori
/note="SV40 origin of replication"
rep_origin 9169..9624
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
ORIGIN
1 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
61 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
661 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
1021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
1081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
1141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
1201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
1261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
1321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
1381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
1441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
1501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
1561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
1621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
1681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
1741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
1801 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
1861 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
1921 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
1981 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
2041 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
2101 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt
2161 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat
2221 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc
2281 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg
2341 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc
2401 gcattgcaga gatattgtat ttaagtgcct agctcgatac aataaacggg tctctctggt
2461 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
2521 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
2581 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
2641 cagggacctg aaagcgaaag ggaaaccaga gctctctcga cgcaggactc ggcttgctga
2701 agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa ttttgactag
2761 cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg ggagaattag
2821 atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata aattaaaaca
2881 tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc tgttagaaac
2941 atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga caggatcaga
3001 agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc aaaggataga
3061 gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca aaagtaagac
3121 caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg agggacaatt
3181 ggagaagtga attatataaa tataaagtag taaaaattga accattagga gtagcaccca
3241 ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata ggagctttgt
3301 tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcctcaatg acgctgacgg
3361 tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg ctgagggcta
3421 ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag ctccaggcaa
3481 gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt tggggttgct
3541 ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt aataaatctc
3601 tggaacagat tggaatcaca cgacctggat ggagtgggac agagaaatta acaattacac
3661 aagcttaata cactccttaa ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga
3721 attattggaa ttagataaat gggcaagttt gtggaattgg tttaacataa caaattggct
3781 gtggtatata aaattattca taatgatagt aggaggcttg gtaggtttaa gaatagtttt
3841 tgctgtactt tctatagtga atagagttag gcagggatat tcaccattat cgtttcagac
3901 ccacctccca accccgaggg gacccgacag gcccgaagga atagaagaag aaggtggaga
3961 gagagacaga gacagatcca ttcgattagt gaacggatct cgacggttaa cttttaaaag
4021 aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa tagcaacaga
4081 catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaatttta tcgataagct
4141 tgggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
4201 ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc
4261 cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg
4321 tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat
4381 tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc
4441 atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt
4501 gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac
4561 caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc
4621 ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa ccgtcagatc
4681 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccgact ctagtccagt
4741 gtggtggaat tctgcagata tcaacaagtt tgtacaaaaa agcaggctcc accatggaag
4801 acgccaaaaa cataaagaaa ggcccggcgc cattctatcc gctggaagat ggaaccgctg
4861 gagagcaact gcataaggct atgaagagat acgccctggt tcctggaaca attgctttta
4921 cagatgcaca tatcgaggtg gacatcactt acgctgagta cttcgaaatg tccgttcggt
4981 tggcagaagc tatgaaacga tatgggctga atacaaatca cagaatcgtc gtatgcagtg
5041 aaaactctct tcaattcttt atgccggtgt tgggcgcgtt atttatcgga gttgcagttg
5101 cgcccgcgaa cgacatttat aatgaacgtg aattgctcaa cagtatgggc atttcgcagc
5161 ctaccgtggt gttcgtttcc aaaaaggggt tgcaaaaaat tttgaacgtg caaaaaaagc
5221 tcccaatcat ccaaaaaatt attatcatgg attctaaaac ggattaccag ggatttcagt
5281 cgatgtacac gttcgtcaca tctcatctac ctcccggttt taatgaatac gattttgtgc
5341 cagagtcctt cgatagggac aagacaattg cactgatcat gaactcctct ggatctactg
5401 gtctgcctaa aggtgtcgct ctgcctcata gaactgcctg cgtgagattc tcgcatgcca
5461 gagatcctat ttttggcaat caaatcattc cggatactgc gattttaagt gttgttccat
5521 tccatcacgg ttttggaatg tttactacac tcggatattt gatatgtgga tttcgagtcg
5581 tcttaatgta tagatttgaa gaagagctgt ttctgaggag ccttcaggat tacaagattc
5641 aaagtgcgct gctggtgcca accctattct ccttcttcgc caaaagcact ctgattgaca
5701 aatacgattt atctaattta cacgaaattg cttctggtgg cgctcccctc tctaaggaag
5761 tcggggaagc ggttgccaag aggttccatc tgccaggtat caggcaagga tatgggctca
5821 ctgagactac atcagctatt ctgattacac ccgaggggga tgataaaccg ggcgcggtcg
5881 gtaaagttgt tccatttttt gaagcgaagg ttgtggatct ggataccggg aaaacgctgg
5941 gcgttaatca aagaggcgaa ctgtgtgtga gaggtcctat gattatgtcc ggttatgtaa
6001 acaatccgga agcgaccaac gccttgattg acaaggatgg atggctacat tctggagaca
6061 tagcttactg ggacgaagac gaacacttct tcatcgttga ccgcctgaag tctctgatta
6121 agtacaaagg ctatcaggtg gctcccgctg aattggaatc catcttgctc caacacccca
6181 acatcttcga cgcaggtgtc gcaggtcttc ccgacgatga cgccggtgaa cttcccgccg
6241 ccgttgttgt tttggagcac ggaaagacga tgacggaaaa agagatcgtg gattacgtcg
6301 ccagtcaagt aacaaccgcg aaaaagttgc gcggaggagt tgtgtttgtg gacgaagtac
6361 cgaaaggtct taccggaaaa ctcgacgcaa gaaaaatcag agagatcctc ataaaggcca
6421 agaagggcgg aaagatcgcc gtgtaattct agacccagct ttcttgtaca aagtggttga
6481 tatccagcac agtggcggcc gctcgacaat caacctctgg attacaaaat ttgtgaaaga
6541 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
6601 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc
6661 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc
6721 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt
6781 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt
6841 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
6901 aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg
6961 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg
7021 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt
7081 tgggccgcct ccccgcctgg aattctaccg ggtaggggag gcgcttttcc caaggcagtc
7141 tggagcatgc gctttagcag ccccgctggg cacttggcgc tacacaagtg gcctctggcc
7201 tcgcacacat tccacatcca ccggtaggcg ccaaccggct ccgttctttg gtggcccctt
7261 cgcgccacct tctactcctc ccctagtcag gaagttcccc cccgccccgc agctcgcgtc
7321 gtgcaggacg tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga tggacagcac
7381 cgctgagcaa tggaagcggg taggcctttg gggcagcggc caatagcagc tttgctcctt
7441 cgctttctgg gctcagaggc tgggaagggg tgggtccggg ggcgggctca ggggcgggct
7501 caggggcggg gcgggcgccc gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa
7561 gcgcacgtct gccgcgctgt tctcctcttc ctcatctccg ggcctttcga cctgcagccc
7621 aagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
7681 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
7741 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
7801 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
7861 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
7921 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
7981 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
8041 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
8101 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
8161 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
8221 ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc acgaccccat
8281 gcatctcgag ggcccggtac ctttaagacc aatgacttac aaggcagctg tagatcttag
8341 ccacttttta aaagaaaagg ggggactgga agggctagct cactcccaac gaagacaaga
8401 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
8461 tggctgccta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
8521 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
8581 agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt atttataact
8641 tgcaaagaaa tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta
8701 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
8761 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc
8821 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
8881 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
8941 ggaggcctag gcttttgcgt cgagacgtac ccaattcgcc ctatagtgag tcgtattacg
9001 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
9061 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
9121 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcgac gcgccctgta
9181 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
9241 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
9301 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
9361 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
9421 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
9481 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
9541 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
9601 acaaaatatt aacgtttaca atttcc
//
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
| P0317/pET28a-TEV蛋白酶基因质粒 |
| P0318/pCaspase切割酶基因质粒 |
| P0322/pGEX-HRV3C①切割酶基因质粒 |
| P0323/pET28a-ULP(SUMO)切割酶基因质粒 |
| P0319/pUC57-T4 DNA Ligase连接酶基因质粒 |
| P1805/pMy-BirA-T2A-eGFP生物素酶基因质粒 |
| P0324/pTTT T载体制备质粒 |
| P0320/pDL2000 DNA Marker制备质粒 |
| P1599/pPSU2 DNA Marker制备质粒 |
| P1109/pFUPI AoT5Cyp猫头鹰猴基因质粒 |
| P0973/pPH37酵母基因质粒 |
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pLenti CMV Puro LUC (w168-1) 慢病毒表达质粒
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