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- 详细信息
- 询价记录
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
gag-pol
- 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：pCMV-dR8.91慢病毒包装质粒
别称: gag-pol
质粒属性
质粒简介
质粒图谱
质粒序列
LOCUS Exported 12150 bp ds-DNA circular SYN 27-AUG-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 31
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12150)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Saturday, August 27, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..12150
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 377..580
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 855..2357
/codon_start=1
/gene="gag"
/product="gag protein from human immunodeficiency virus 1"
/note="HIV-1 gag"
/translation="MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFA
VNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKI
EEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKA
FSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGP
IAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTS
ILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPG
ATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG
HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAP
PEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ"
CDS 2150..5161
/codon_start=1
/gene="pol"
/product="pol protein from human immunodeficiency virus 1"
/note="HIV-1 pol"
/translation="FFREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAG
ADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIG
GIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETV
PVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPVFAIKKKD
STKWRKLVDFRELNKRTQDFWEVQLGIP-HPAGLKQKKSVTVLDVGDAYFSVPLDKDFRK
YTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCSMTKILEPFRKQNPDIVIYQYMD
DLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPI
VLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVRQLCKLLRGTKALTEVVPLTEEAELEL
AENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMKGAHT
NDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPP
LVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPLTDTTNQKTELQ
AIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAH
KGIGGNEQVDKLVSAGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVA
SCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQET
AYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNK
ELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQK
QITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQ
MAGDDCVASRQDED"
misc_feature 4846..4963
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
misc_feature 5735..5968
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
promoter complement(7140..7158)
/note="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
promoter 7946..8050
/gene="bla"
/note="AmpR promoter"
CDS 8051..8911
/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"
rep_origin 9082..9670
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 9916..10245
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 10096..10231
/note="SV40 ori"
/note="SV40 origin of replication"
intron 11478..11543
/note="small t intron"
/note="simian virus 40 (SV40) small t antigen intron"
CDS 11673..11693
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
enhancer 12147..376
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
ORIGIN
1 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
61 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
121 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
181 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
241 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
301 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
361 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
421 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
481 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
541 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat
601 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
661 cctccgcggc cgggaacggt gcattggaac gcggattccc cgtgccaaga gtgacgtaag
721 taccgcctat agagtctata ggcccacccc cttggcttct tatgcgacgg atcgatcccg
781 taataagctt cgaggtccgc ggccggccgc gttgacgcgc acggcaagag gcgaggggcg
841 gcgactggtg agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgat
901 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
961 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
1021 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
1081 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
1141 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag aaaaaagcac
1201 agcaagcagc agctgacaca ggacacagca atcaggtcag ccaaaattac cctatagtgc
1261 agaacatcca ggggcaaatg gtacatcagg ccatatcacc tagaacttta aatgcatggg
1321 taaaagtagt agaagagaag gctttcagcc cagaagtgat acccatgttt tcagcattat
1381 cagaaggagc caccccacaa gatttaaaca ccatgctaaa cacagtgggg ggacatcaag
1441 cagccatgca aatgttaaaa gagaccatca atgaggaagc tgcagaatgg gatagagtgc
1501 atccagtgca tgcagggcct attgcaccag gccagatgag agaaccaagg ggaagtgaca
1561 tagcaggaac tactagtacc cttcaggaac aaataggatg gatgacacat aatccaccta
1621 tcccagtagg agaaatctat aaaagatgga taatcctggg attaaataaa atagtaagaa
1681 tgtatagccc taccagcatt ctggacataa gacaaggacc aaaggaaccc tttagagact
1741 atgtagaccg attctataaa actctaagag ccgagcaagc ttcacaagag gtaaaaaatt
1801 ggatgacaga aaccttgttg gtccaaaatg cgaacccaga ttgtaagact attttaaaag
1861 cattgggacc aggagcgaca ctagaagaaa tgatgacagc atgtcaggga gtggggggac
1921 ccggccataa agcaagagtt ttggctgaag caatgagcca agtaacaaat ccagctacca
1981 taatgataca gaaaggcaat tttaggaacc aaagaaagac tgttaagtgt ttcaattgtg
2041 gcaaagaagg gcacatagcc aaaaattgca gggcccctag gaaaaagggc tgttggaaat
2101 gtggaaagga aggacaccaa atgaaagatt gtactgagag acaggctaat tttttaggga
2161 agatctggcc ttcccacaag ggaaggccag ggaattttct tcagagcaga ccagagccaa
2221 cagccccacc agaagagagc ttcaggtttg gggaagagac aacaactccc tctcagaagc
2281 aggagccgat agacaaggaa ctgtatcctt tagcttccct cagatcactc tttggcagcg
2341 acccctcgtc acaataaaga taggggggca attaaaggaa gctctattag atacaggagc
2401 agatgataca gtattagaag aaatgaattt gccaggaaga tggaaaccaa aaatgatagg
2461 gggaattgga ggttttatca aagtaagaca gtatgatcag atactcatag aaatctgcgg
2521 acataaagct ataggtacag tattagtagg acctacacct gtcaacataa ttggaagaaa
2581 tctgttgact cagattggct gcactttaaa ttttcccatt agtcctattg agactgtacc
2641 agtaaaatta aagccaggaa tggatggccc aaaagttaaa caatggccat tgacagaaga
2701 aaaaataaaa gcattagtag aaatttgtac agaaatggaa aaggaaggaa aaatttcaaa
2761 aattgggcct gaaaatccat acaatactcc agtatttgcc ataaagaaaa aagacagtac
2821 taaatggaga aaattagtag atttcagaga acttaataag agaactcaag atttctggga
2881 agttcaatta ggaataccac atcctgcagg gttaaaacag aaaaaatcag taacagtact
2941 ggatgtgggc gatgcatatt tttcagttcc cttagataaa gacttcagga agtatactgc
3001 atttaccata cctagtataa acaatgagac accagggatt agatatcagt acaatgtgct
3061 tccacaggga tggaaaggat caccagcaat attccagtgt agcatgacaa aaatcttaga
3121 gccttttaga aaacaaaatc cagacatagt catctatcaa tacatggatg atttgtatgt
3181 aggatctgac ttagaaatag ggcagcatag aacaaaaata gaggaactga gacaacatct
3241 gttgaggtgg ggatttacca caccagacaa aaaacatcag aaagaacctc cattcctttg
3301 gatgggttat gaactccatc ctgataaatg gacagtacag cctatagtgc tgccagaaaa
3361 ggacagctgg actgtcaatg acatacagaa attagtggga aaattgaatt gggcaagtca
3421 gatttatgca gggattaaag taaggcaatt atgtaaactt cttaggggaa ccaaagcact
3481 aacagaagta gtaccactaa cagaagaagc agagctagaa ctggcagaaa acagggagat
3541 tctaaaagaa ccggtacatg gagtgtatta tgacccatca aaagacttaa tagcagaaat
3601 acagaagcag gggcaaggcc aatggacata tcaaatttat caagagccat ttaaaaatct
3661 gaaaacagga aagtatgcaa gaatgaaggg tgcccacact aatgatgtga aacaattaac
3721 agaggcagta caaaaaatag ccacagaaag catagtaata tggggaaaga ctcctaaatt
3781 taaattaccc atacaaaagg aaacatggga agcatggtgg acagagtatt ggcaagccac
3841 ctggattcct gagtgggagt ttgtcaatac ccctccctta gtgaagttat ggtaccagtt
3901 agagaaagaa cccataatag gagcagaaac tttctatgta gatggggcag ccaataggga
3961 aactaaatta ggaaaagcag gatatgtaac tgacagagga agacaaaaag ttgtccccct
4021 aacggacaca acaaatcaga agactgagtt acaagcaatt catctagctt tgcaggattc
4081 gggattagaa gtaaacatag tgacagactc acaatatgca ttgggaatca ttcaagcaca
4141 accagataag agtgaatcag agttagtcag tcaaataata gagcagttaa taaaaaagga
4201 aaaagtctac ctggcatggg taccagcaca caaaggaatt ggaggaaatg aacaagtaga
4261 taaattggtc agtgctggaa tcaggaaagt actattttta gatggaatag ataaggccca
4321 agaagaacat gagaaatatc acagtaattg gagagcaatg gctagtgatt ttaacctacc
4381 acctgtagta gcaaaagaaa tagtagccag ctgtgataaa tgtcagctaa aaggggaagc
4441 catgcatgga caagtagact gtagcccagg aatatggcag ctagattgta cacatttaga
4501 aggaaaagtt atcttggtag cagttcatgt agccagtgga tatatagaag cagaagtaat
4561 tccagcagag acagggcaag aaacagcata cttcctctta aaattagcag gaagatggcc
4621 agtaaaaaca gtacatacag acaatggcag caatttcacc agtactacag ttaaggccgc
4681 ctgttggtgg gcggggatca agcaggaatt tggcattccc tacaatcccc aaagtcaagg
4741 agtaatagaa tctatgaata aagaattaaa gaaaattata ggacaggtaa gagatcaggc
4801 tgaacatctt aagacagcag tacaaatggc agtattcatc cacaatttta aaagaaaagg
4861 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
4921 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
4981 cagcagagat ccagtttgga aaggaccagc aaagctcctc tggaaaggtg aaggggcagt
5041 agtaatacaa gataatagtg acataaaagt agtgccaaga agaaaagcaa agatcatcag
5101 ggattatgga aaacagatgg caggtgatga ttgtgtggca agtagacagg atgaggatta
5161 acacatggaa ttctgcaaca actgctgttt atccatttca gaattgggtg tcgacatagc
5221 agaataggcg ttactcgaca gaggagagca agaaatggag ccagtagatc ctagactaga
5281 gccctggaag catccaggaa gtcagcctaa aactgcttgt accaattgct attgtaaaaa
5341 gtgttgcttt cattgccaag tttgtttcat gacaaaagcc ttaggcatct cctatggcag
5401 gaagaagcgg agacagcgac gaagagctca tcagaacagt cagactcatc aagcttctct
5461 atcaaagcag taagtagtac atgtaatgca acctataata gtagcaatag tagcattagt
5521 agtagcaata ataatagcaa tagttgtgtg gtccatagta atcatagaat ataggaaaat
5581 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
5641 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
5701 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
5761 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
5821 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
5881 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
5941 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
6001 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
6061 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
6121 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
6181 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
6241 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
6301 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
6361 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
6421 gatccattcg attagtgaac ggatccttgg cacttatctg ggacgatctg cggagcctgt
6481 gcctcttcag ctaccaccgc ttgagagact tactcttgat tgtaacgagg attgtggaac
6541 ttctgggacg cagggggtgg gaagccctca aatattggtg gaatctccta caatattgga
6601 gtcaggagct aaagaatagt gctgttagct tgctcaatgc cacagccata gcagtagctg
6661 aggggacaga tagggttata gaagtagtac aaggagcttg tagagctatt cgccacatac
6721 ctagaagaat aagacagggc ttggaaagga ttttgctata agctcgaggc cgccccggtg
6781 accttcagac cttggcactg gaggtggccc ggcagaagcg cggcatcgtg gatcagtgct
6841 gcaccagcat ctgctctctc taccaactgg agaactactg caactaggcc caccactacc
6901 ctgtccaccc ctctgcaatg aataaaacct ttgaaagagc actacaagtt gtgtgtacat
6961 gcgtgcatgt gcatatgtgg tgcgggggga acatgagtgg ggctggctgg agtggcgatg
7021 ataagctgtc aaacatgaga attaattctt gaagacgaaa gggcctcgtg atacgcctat
7081 ttttataggt taatgtcatg ataataatgg tttcttagtc tagaattaat tccgtgtatt
7141 ctatagtgtc acctaaatcg tatgtgtatg atacataagg ttatgtatta attgtagccg
7201 cgttctaacg acaatatgta caagcctaat tgtgtagcat ctggcttact gaagcagacc
7261 ctatcatctc tctcgtaaac tgccgtcaga gtcggtttgg ttggacgaac cttctgagtt
7321 tctggtaacg ccgtcccgca cccggaaatg gtcagcgaac caatcagcag ggtcatcgct
7381 agccagatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
7441 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
7501 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
7561 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
7621 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaatggt gcactctcag
7681 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
7741 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
7801 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
7861 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
7921 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
7981 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
8041 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
8101 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
8161 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
8221 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
8281 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
8341 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
8401 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
8461 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
8521 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
8581 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
8641 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
8701 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
8761 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
8821 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
8881 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
8941 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
9001 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
9061 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
9121 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
9181 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
9241 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
9301 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
9361 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
9421 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
9481 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
9541 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
9601 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
9661 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
9721 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
9781 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
9841 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
9901 atgcagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
9961 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
10021 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
10081 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
10141 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
10201 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct tggacacaag
10261 acaggcttgc gagatatgtt tgagaatacc actttatccc gcgtcaggga gaggcagtgc
10321 gtaaaaagac gcggactcat gtgaaatact ggtttttagt gcgccagatc tctataatct
10381 cgcgcaacct attttcccct cgaacacttt ttaagccgta gataaacagg ctgggacact
10441 tcacatgagc gaaaaataca tcgtcacctg ggacatgttg cagatccatg cacgtaaact
10501 cgcaagccga ctgatgcctt ctgaacaatg gaaaggcatt attgccgtaa gccgtggcgg
10561 tctgtaccgg gtgcgttact ggcgcgtgaa ctgggtattc gtcatgtcga taccgtttgt
10621 atttccagct acgatcacga caaccagcgc gagcttaaag tgctgaaacg cgcagaaggc
10681 gatggcgaag gcttcatcgt tattgatgac ctggtggata ccggtggtac tgcggttgcg
10741 attcgtgaaa tgtatccaaa agcgcacttt gtcaccatct tcgcaaaacc ggctggtcgt
10801 ccgctggttg atgactatgt tgttgatatc ccgcaagata cctggattga acagccgtgg
10861 gatatgggcg tcgtattcgt cccgccaatc tccggtcgct aatcttttca acgcctggca
10921 ctgccgggcg ttgttctttt taacttcagg cgggttacaa tagtttccag taagtattct
10981 ggaggctgca tccatgacac aggcaaacct gagcgaaacc ctgttcaaac cccgctttaa
11041 acatcctgaa acctcgacgc tagtccgccg ctttaatcac ggcgcacaac cgcctgtgca
11101 gtcggccctt gatggtaaaa ccatccctca ctggtatcgc atgattaacc gtctgatgtg
11161 gatctggcgc ggcattgacc cacgcgaaat cctcgacgtc caggcacgta ttgtgatgag
11221 cgatgccgaa cgtaccgacg atgatttata cgatacggtg attggctacc gtggcggcaa
11281 ctggatttat gagtgggccc cggatctttg tgaaggaacc ttacttctgt ggtgtgacat
11341 aattggacaa actacctaca gagatttaaa gctctaaggt aaatataaaa tttttaaccc
11401 ggatctttgt gaaggaacct tacttctgtg gtgtgacata attggacaaa ctacctacag
11461 agatttaaag ctctaaggta aatataaaat ttttaagtgt ataatgtgtt aaactactga
11521 ttctaattgt ttgtgtattt tagattccaa cctatggaac tgatgaatgg gagcagtggt
11581 ggaatgcctt taatgaggaa aacctgtttt gctcagaaga aatgccatct agtgatgatg
11641 aggctactgc tgactctcaa cattctactc ctccaaaaaa gaagagaaag gtagaagacc
11701 ccaaggactt tccttcagaa ttgctaagtt ttttgagtca tgctgtgttt agtaatagaa
11761 ctcttgcttg ctttgctatt tacaccacaa aggaaaaagc tgcactgcta tacaagaaaa
11821 ttatggaaaa atattctgta acctttataa gtaggcataa cagttataat cataacatac
11881 tgttttttct tactccacac aggcatagag tgtctgctat taataactat gctcaaaaat
11941 tgtgtacctt tagcttttta atttgtaaag gggttaataa ggaatatttg atgtatagtg
12001 ccttgactag agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
12061 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttggg
12121 ctgcaggaat taattcgagc tcgcccgaca
//
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：pCMV-dR8.91慢病毒包装质粒
别称: gag-pol
| 启动子: | CMV |
|---|---|
| 复制子: | pUC |
| 质粒分类: | 病毒系列，慢病毒包装载体 |
| 质粒大小: | 12150bp |
| 原核抗性: | Amp |
| 克隆菌株: | DH5a |
| 培养条件: | 37度 |
| 表达宿主: | 哺乳细胞 |
| 诱导方式: | 无须诱导，瞬时表达 |
| 5'测序引物: | SP6：ATTTAGGTGACACTATAGAA |
| 3'测序引物: | 根据序列设计引物 |
质粒属性
| 载体宿主： | 哺乳细胞,慢病毒 |
|---|---|
| 载体用途： | 包装辅助 |
| 基因种属： | 病毒 |
| 基因类型： | ORF |
| 原核抗性： | Amp |
| 筛选标记： | |
| 荧光蛋白： |
质粒简介
质粒图谱
质粒序列
LOCUS Exported 12150 bp ds-DNA circular SYN 27-AUG-2016
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS Untitled 31
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12150)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Saturday, August 27, 2016 from SnapGene Viewer 3.1.4
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..12150
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 377..580
/note="CMV promoter"
/note="human cytomegalovirus (CMV) immediate early
promoter"
CDS 855..2357
/codon_start=1
/gene="gag"
/product="gag protein from human immunodeficiency virus 1"
/note="HIV-1 gag"
/translation="MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFA
VNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKI
EEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKA
FSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGP
IAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTS
ILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPG
ATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEG
HIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAP
PEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ"
CDS 2150..5161
/codon_start=1
/gene="pol"
/product="pol protein from human immunodeficiency virus 1"
/note="HIV-1 pol"
/translation="FFREDLAFPQGKAREFSSEQTRANSPTRRELQVWGRDNNSLSEAG
ADRQGTVSFSFPQITLWQRPLVTIKIGGQLKEALLDTGADDTVLEEMNLPGRWKPKMIG
GIGGFIKVRQYDQILIEICGHKAIGTVLVGPTPVNIIGRNLLTQIGCTLNFPISPIETV
PVKLKPGMDGPKVKQWPLTEEKIKALVEICTEMEKEGKISKIGPENPYNTPVFAIKKKD
STKWRKLVDFRELNKRTQDFWEVQLGIP-HPAGLKQKKSVTVLDVGDAYFSVPLDKDFRK
YTAFTIPSINNETPGIRYQYNVLPQGWKGSPAIFQCSMTKILEPFRKQNPDIVIYQYMD
DLYVGSDLEIGQHRTKIEELRQHLLRWGFTTPDKKHQKEPPFLWMGYELHPDKWTVQPI
VLPEKDSWTVNDIQKLVGKLNWASQIYAGIKVRQLCKLLRGTKALTEVVPLTEEAELEL
AENREILKEPVHGVYYDPSKDLIAEIQKQGQGQWTYQIYQEPFKNLKTGKYARMKGAHT
NDVKQLTEAVQKIATESIVIWGKTPKFKLPIQKETWEAWWTEYWQATWIPEWEFVNTPP
LVKLWYQLEKEPIIGAETFYVDGAANRETKLGKAGYVTDRGRQKVVPLTDTTNQKTELQ
AIHLALQDSGLEVNIVTDSQYALGIIQAQPDKSESELVSQIIEQLIKKEKVYLAWVPAH
KGIGGNEQVDKLVSAGIRKVLFLDGIDKAQEEHEKYHSNWRAMASDFNLPPVVAKEIVA
SCDKCQLKGEAMHGQVDCSPGIWQLDCTHLEGKVILVAVHVASGYIEAEVIPAETGQET
AYFLLKLAGRWPVKTVHTDNGSNFTSTTVKAACWWAGIKQEFGIPYNPQSQGVIESMNK
ELKKIIGQVRDQAEHLKTAVQMAVFIHNFKRKGGIGGYSAGERIVDIIATDIQTKELQK
QITKIQNFRVYYRDSRDPVWKGPAKLLWKGEGAVVIQDNSDIKVVPRRKAKIIRDYGKQ
MAGDDCVASRQDED"
misc_feature 4846..4963
/note="cPPT/CTS"
/note="central polypurine tract and central termination
sequence of HIV-1"
misc_feature 5735..5968
/note="RRE"
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
promoter complement(7140..7158)
/note="SP6 promoter"
/note="promoter for bacteriophage SP6 RNA polymerase"
promoter 7946..8050
/gene="bla"
/note="AmpR promoter"
CDS 8051..8911
/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"
rep_origin 9082..9670
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
promoter 9916..10245
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 10096..10231
/note="SV40 ori"
/note="SV40 origin of replication"
intron 11478..11543
/note="small t intron"
/note="simian virus 40 (SV40) small t antigen intron"
CDS 11673..11693
/codon_start=1
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/translation="PKKKRKV"
enhancer 12147..376
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
ORIGIN
1 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
61 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
121 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
181 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
241 gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca
301 ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt
361 catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt
421 tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca
481 ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg
541 cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga accgtcagat
601 cgcctggaga cgccatccac gctgttttga cctccataga agacaccggg accgatccag
661 cctccgcggc cgggaacggt gcattggaac gcggattccc cgtgccaaga gtgacgtaag
721 taccgcctat agagtctata ggcccacccc cttggcttct tatgcgacgg atcgatcccg
781 taataagctt cgaggtccgc ggccggccgc gttgacgcgc acggcaagag gcgaggggcg
841 gcgactggtg agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgat
901 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
961 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
1021 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
1081 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
1141 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag aaaaaagcac
1201 agcaagcagc agctgacaca ggacacagca atcaggtcag ccaaaattac cctatagtgc
1261 agaacatcca ggggcaaatg gtacatcagg ccatatcacc tagaacttta aatgcatggg
1321 taaaagtagt agaagagaag gctttcagcc cagaagtgat acccatgttt tcagcattat
1381 cagaaggagc caccccacaa gatttaaaca ccatgctaaa cacagtgggg ggacatcaag
1441 cagccatgca aatgttaaaa gagaccatca atgaggaagc tgcagaatgg gatagagtgc
1501 atccagtgca tgcagggcct attgcaccag gccagatgag agaaccaagg ggaagtgaca
1561 tagcaggaac tactagtacc cttcaggaac aaataggatg gatgacacat aatccaccta
1621 tcccagtagg agaaatctat aaaagatgga taatcctggg attaaataaa atagtaagaa
1681 tgtatagccc taccagcatt ctggacataa gacaaggacc aaaggaaccc tttagagact
1741 atgtagaccg attctataaa actctaagag ccgagcaagc ttcacaagag gtaaaaaatt
1801 ggatgacaga aaccttgttg gtccaaaatg cgaacccaga ttgtaagact attttaaaag
1861 cattgggacc aggagcgaca ctagaagaaa tgatgacagc atgtcaggga gtggggggac
1921 ccggccataa agcaagagtt ttggctgaag caatgagcca agtaacaaat ccagctacca
1981 taatgataca gaaaggcaat tttaggaacc aaagaaagac tgttaagtgt ttcaattgtg
2041 gcaaagaagg gcacatagcc aaaaattgca gggcccctag gaaaaagggc tgttggaaat
2101 gtggaaagga aggacaccaa atgaaagatt gtactgagag acaggctaat tttttaggga
2161 agatctggcc ttcccacaag ggaaggccag ggaattttct tcagagcaga ccagagccaa
2221 cagccccacc agaagagagc ttcaggtttg gggaagagac aacaactccc tctcagaagc
2281 aggagccgat agacaaggaa ctgtatcctt tagcttccct cagatcactc tttggcagcg
2341 acccctcgtc acaataaaga taggggggca attaaaggaa gctctattag atacaggagc
2401 agatgataca gtattagaag aaatgaattt gccaggaaga tggaaaccaa aaatgatagg
2461 gggaattgga ggttttatca aagtaagaca gtatgatcag atactcatag aaatctgcgg
2521 acataaagct ataggtacag tattagtagg acctacacct gtcaacataa ttggaagaaa
2581 tctgttgact cagattggct gcactttaaa ttttcccatt agtcctattg agactgtacc
2641 agtaaaatta aagccaggaa tggatggccc aaaagttaaa caatggccat tgacagaaga
2701 aaaaataaaa gcattagtag aaatttgtac agaaatggaa aaggaaggaa aaatttcaaa
2761 aattgggcct gaaaatccat acaatactcc agtatttgcc ataaagaaaa aagacagtac
2821 taaatggaga aaattagtag atttcagaga acttaataag agaactcaag atttctggga
2881 agttcaatta ggaataccac atcctgcagg gttaaaacag aaaaaatcag taacagtact
2941 ggatgtgggc gatgcatatt tttcagttcc cttagataaa gacttcagga agtatactgc
3001 atttaccata cctagtataa acaatgagac accagggatt agatatcagt acaatgtgct
3061 tccacaggga tggaaaggat caccagcaat attccagtgt agcatgacaa aaatcttaga
3121 gccttttaga aaacaaaatc cagacatagt catctatcaa tacatggatg atttgtatgt
3181 aggatctgac ttagaaatag ggcagcatag aacaaaaata gaggaactga gacaacatct
3241 gttgaggtgg ggatttacca caccagacaa aaaacatcag aaagaacctc cattcctttg
3301 gatgggttat gaactccatc ctgataaatg gacagtacag cctatagtgc tgccagaaaa
3361 ggacagctgg actgtcaatg acatacagaa attagtggga aaattgaatt gggcaagtca
3421 gatttatgca gggattaaag taaggcaatt atgtaaactt cttaggggaa ccaaagcact
3481 aacagaagta gtaccactaa cagaagaagc agagctagaa ctggcagaaa acagggagat
3541 tctaaaagaa ccggtacatg gagtgtatta tgacccatca aaagacttaa tagcagaaat
3601 acagaagcag gggcaaggcc aatggacata tcaaatttat caagagccat ttaaaaatct
3661 gaaaacagga aagtatgcaa gaatgaaggg tgcccacact aatgatgtga aacaattaac
3721 agaggcagta caaaaaatag ccacagaaag catagtaata tggggaaaga ctcctaaatt
3781 taaattaccc atacaaaagg aaacatggga agcatggtgg acagagtatt ggcaagccac
3841 ctggattcct gagtgggagt ttgtcaatac ccctccctta gtgaagttat ggtaccagtt
3901 agagaaagaa cccataatag gagcagaaac tttctatgta gatggggcag ccaataggga
3961 aactaaatta ggaaaagcag gatatgtaac tgacagagga agacaaaaag ttgtccccct
4021 aacggacaca acaaatcaga agactgagtt acaagcaatt catctagctt tgcaggattc
4081 gggattagaa gtaaacatag tgacagactc acaatatgca ttgggaatca ttcaagcaca
4141 accagataag agtgaatcag agttagtcag tcaaataata gagcagttaa taaaaaagga
4201 aaaagtctac ctggcatggg taccagcaca caaaggaatt ggaggaaatg aacaagtaga
4261 taaattggtc agtgctggaa tcaggaaagt actattttta gatggaatag ataaggccca
4321 agaagaacat gagaaatatc acagtaattg gagagcaatg gctagtgatt ttaacctacc
4381 acctgtagta gcaaaagaaa tagtagccag ctgtgataaa tgtcagctaa aaggggaagc
4441 catgcatgga caagtagact gtagcccagg aatatggcag ctagattgta cacatttaga
4501 aggaaaagtt atcttggtag cagttcatgt agccagtgga tatatagaag cagaagtaat
4561 tccagcagag acagggcaag aaacagcata cttcctctta aaattagcag gaagatggcc
4621 agtaaaaaca gtacatacag acaatggcag caatttcacc agtactacag ttaaggccgc
4681 ctgttggtgg gcggggatca agcaggaatt tggcattccc tacaatcccc aaagtcaagg
4741 agtaatagaa tctatgaata aagaattaaa gaaaattata ggacaggtaa gagatcaggc
4801 tgaacatctt aagacagcag tacaaatggc agtattcatc cacaatttta aaagaaaagg
4861 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
4921 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
4981 cagcagagat ccagtttgga aaggaccagc aaagctcctc tggaaaggtg aaggggcagt
5041 agtaatacaa gataatagtg acataaaagt agtgccaaga agaaaagcaa agatcatcag
5101 ggattatgga aaacagatgg caggtgatga ttgtgtggca agtagacagg atgaggatta
5161 acacatggaa ttctgcaaca actgctgttt atccatttca gaattgggtg tcgacatagc
5221 agaataggcg ttactcgaca gaggagagca agaaatggag ccagtagatc ctagactaga
5281 gccctggaag catccaggaa gtcagcctaa aactgcttgt accaattgct attgtaaaaa
5341 gtgttgcttt cattgccaag tttgtttcat gacaaaagcc ttaggcatct cctatggcag
5401 gaagaagcgg agacagcgac gaagagctca tcagaacagt cagactcatc aagcttctct
5461 atcaaagcag taagtagtac atgtaatgca acctataata gtagcaatag tagcattagt
5521 agtagcaata ataatagcaa tagttgtgtg gtccatagta atcatagaat ataggaaaat
5581 ggccgctgat cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat
5641 ataaatataa agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa
5701 gagtggtgca gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg
5761 gagcagcagg aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat
5821 tattgtctgg tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc
5881 atctgttgca actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg
5941 aaagatacct aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt
6001 gcaccactgc tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga
6061 atcacacgac ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact
6121 ccttaattga agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag
6181 ataaatgggc aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat
6241 tattcataat gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta
6301 tagtgaatag agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc
6361 cgaggggacc cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca
6421 gatccattcg attagtgaac ggatccttgg cacttatctg ggacgatctg cggagcctgt
6481 gcctcttcag ctaccaccgc ttgagagact tactcttgat tgtaacgagg attgtggaac
6541 ttctgggacg cagggggtgg gaagccctca aatattggtg gaatctccta caatattgga
6601 gtcaggagct aaagaatagt gctgttagct tgctcaatgc cacagccata gcagtagctg
6661 aggggacaga tagggttata gaagtagtac aaggagcttg tagagctatt cgccacatac
6721 ctagaagaat aagacagggc ttggaaagga ttttgctata agctcgaggc cgccccggtg
6781 accttcagac cttggcactg gaggtggccc ggcagaagcg cggcatcgtg gatcagtgct
6841 gcaccagcat ctgctctctc taccaactgg agaactactg caactaggcc caccactacc
6901 ctgtccaccc ctctgcaatg aataaaacct ttgaaagagc actacaagtt gtgtgtacat
6961 gcgtgcatgt gcatatgtgg tgcgggggga acatgagtgg ggctggctgg agtggcgatg
7021 ataagctgtc aaacatgaga attaattctt gaagacgaaa gggcctcgtg atacgcctat
7081 ttttataggt taatgtcatg ataataatgg tttcttagtc tagaattaat tccgtgtatt
7141 ctatagtgtc acctaaatcg tatgtgtatg atacataagg ttatgtatta attgtagccg
7201 cgttctaacg acaatatgta caagcctaat tgtgtagcat ctggcttact gaagcagacc
7261 ctatcatctc tctcgtaaac tgccgtcaga gtcggtttgg ttggacgaac cttctgagtt
7321 tctggtaacg ccgtcccgca cccggaaatg gtcagcgaac caatcagcag ggtcatcgct
7381 agccagatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac aggtgcggtt
7441 gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca cttcgggctc
7501 atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg actgttgggc
7561 gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct caacctacta
7621 ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaatggt gcactctcag
7681 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
7741 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
7801 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
7861 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
7921 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
7981 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
8041 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
8101 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
8161 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
8221 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
8281 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
8341 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
8401 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
8461 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
8521 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
8581 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
8641 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
8701 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
8761 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
8821 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
8881 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
8941 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
9001 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
9061 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
9121 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
9181 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
9241 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
9301 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
9361 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
9421 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
9481 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
9541 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
9601 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
9661 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
9721 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
9781 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
9841 ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta
9901 atgcagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
9961 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
10021 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
10081 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
10141 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
10201 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct tggacacaag
10261 acaggcttgc gagatatgtt tgagaatacc actttatccc gcgtcaggga gaggcagtgc
10321 gtaaaaagac gcggactcat gtgaaatact ggtttttagt gcgccagatc tctataatct
10381 cgcgcaacct attttcccct cgaacacttt ttaagccgta gataaacagg ctgggacact
10441 tcacatgagc gaaaaataca tcgtcacctg ggacatgttg cagatccatg cacgtaaact
10501 cgcaagccga ctgatgcctt ctgaacaatg gaaaggcatt attgccgtaa gccgtggcgg
10561 tctgtaccgg gtgcgttact ggcgcgtgaa ctgggtattc gtcatgtcga taccgtttgt
10621 atttccagct acgatcacga caaccagcgc gagcttaaag tgctgaaacg cgcagaaggc
10681 gatggcgaag gcttcatcgt tattgatgac ctggtggata ccggtggtac tgcggttgcg
10741 attcgtgaaa tgtatccaaa agcgcacttt gtcaccatct tcgcaaaacc ggctggtcgt
10801 ccgctggttg atgactatgt tgttgatatc ccgcaagata cctggattga acagccgtgg
10861 gatatgggcg tcgtattcgt cccgccaatc tccggtcgct aatcttttca acgcctggca
10921 ctgccgggcg ttgttctttt taacttcagg cgggttacaa tagtttccag taagtattct
10981 ggaggctgca tccatgacac aggcaaacct gagcgaaacc ctgttcaaac cccgctttaa
11041 acatcctgaa acctcgacgc tagtccgccg ctttaatcac ggcgcacaac cgcctgtgca
11101 gtcggccctt gatggtaaaa ccatccctca ctggtatcgc atgattaacc gtctgatgtg
11161 gatctggcgc ggcattgacc cacgcgaaat cctcgacgtc caggcacgta ttgtgatgag
11221 cgatgccgaa cgtaccgacg atgatttata cgatacggtg attggctacc gtggcggcaa
11281 ctggatttat gagtgggccc cggatctttg tgaaggaacc ttacttctgt ggtgtgacat
11341 aattggacaa actacctaca gagatttaaa gctctaaggt aaatataaaa tttttaaccc
11401 ggatctttgt gaaggaacct tacttctgtg gtgtgacata attggacaaa ctacctacag
11461 agatttaaag ctctaaggta aatataaaat ttttaagtgt ataatgtgtt aaactactga
11521 ttctaattgt ttgtgtattt tagattccaa cctatggaac tgatgaatgg gagcagtggt
11581 ggaatgcctt taatgaggaa aacctgtttt gctcagaaga aatgccatct agtgatgatg
11641 aggctactgc tgactctcaa cattctactc ctccaaaaaa gaagagaaag gtagaagacc
11701 ccaaggactt tccttcagaa ttgctaagtt ttttgagtca tgctgtgttt agtaatagaa
11761 ctcttgcttg ctttgctatt tacaccacaa aggaaaaagc tgcactgcta tacaagaaaa
11821 ttatggaaaa atattctgta acctttataa gtaggcataa cagttataat cataacatac
11881 tgttttttct tactccacac aggcatagag tgtctgctat taataactat gctcaaaaat
11941 tgtgtacctt tagcttttta atttgtaaag gggttaataa ggaatatttg atgtatagtg
12001 ccttgactag agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
12061 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttggg
12121 ctgcaggaat taattcgagc tcgcccgaca
//
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
| P1594/ptdTomato-C1哺乳荧光质粒 |
| P0475/pmCherry-N1哺乳荧光质粒 |
| P0151/pmCherry-C1哺乳荧光质粒 |
| P0152/pDsRed-Monomer-N1哺乳荧光质粒 |
| P0332/pDsRed-Express2-N1哺乳荧光质粒 |
| P0717/PA-mCherry1-C1哺乳荧光质粒 |
| P0718/PA-mCherry1-N1哺乳荧光质粒 |
| P0869/pDsRed1-C1哺乳荧光质粒 |
| P0918/pmScarlet-C1哺乳荧光质粒 |
| P0652/pCAG-DsRed1哺乳荧光质粒 |
| P0715/pmGeos-M-C1哺乳荧光质粒 |
| P0716/pmGeos-M-N1哺乳荧光质粒 |
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文献和实验相关实验
一、材料和试剂 1. Hairpin-pLKO.1vector（OpenBiosystems） 2. Packagingvectors:第二代包装质粒包含gag,pol,和rev基因（pCMV-dR8.91或pCMV-dR8.74psPAX2）和包被质粒（pMD2.G）（Addgene） 3. 脂质体2000（Invitrogen） 4.OptiMEM无血清培养基（Invitrogen） 5. 293T包装细胞（ATCC）
特异性更高。您可以根据实验设计需要进行选择。 2、慢病毒包装载体：Cas9 慢病毒表达载体采用 3 质粒慢病毒系统。您可使用我公司的 pH1、pH2；addgene 载体 psPAX2、pMD2.G 或者 pCMV-dR8.2 dvpr、pCMV-VSVG 均可包装成功。 3、无内毒素质粒提取试剂盒。使用质量可靠的质粒提取试剂盒可以提供更高的转染效率和病毒包装效率。我们采用 Qiagen 的 Plasmid Plus 系列产品可以取得满意的包装效果。 4、转染
-dR8.91 病毒质粒 pLJM1 病毒质粒 http://www.addgene.org/19319/ pSPax2 病毒质粒 Amp 包装质粒 pMD2.G 病毒
pCMV-dR8.91慢病毒包装质粒
￥100 - 1000
