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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#52961;LentiCRISPR V2
- 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：pLentiCRISPR V2哺乳编辑质粒
别称：Plasmid#52961;LentiCRISPR V2
质粒属性
质粒简介
plentiCRISPR V2哺乳编辑质粒
质粒图谱
质粒序列
LOCUS Exported 14873 bp ds-DNA circular SYN 24-JUL-2019
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14873)
AUTHORS Triple Threat
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..14873
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 238..617
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 618..821
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/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 2440..2557
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2608..2848
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 2858..4737
/label=Stuffer
misc_RNA 4738..4813
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 4875..5086
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 5105..5114
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 5111..9214
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLS-DAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLS-DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 9215..9262
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 9263..9286
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 9296..9352
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 9353..9949
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 9965..10553
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 10625..10858
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 10890..11114
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 11160..11588
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 11602..11931
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 11782..11917
/label=SV40 ori
/note="SV40 origin of replication"
promoter 11979..12026
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 12045..12419
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 12549..12670
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(13121..13709)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13880..14740)
/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"
promoter complement(14741..14845)
/gene="bla"
/label=AmpR promoter
BASE COUNT 3920 a 3699 c 3707 g 3546 t
ORIGIN
1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
2821 ttggctttat atatcttgtg gaaaggacga aacaccggag acggttgtaa atgagcacac
2881 aaaatacaca tgctaaaata ttatattcta tgacctttat aaaatcaacc aaaatcttct
2941 ttttaataac tttagtatca ataattagaa tttttatgtt cctttttgca aacttttaat
3001 aaaaatgagc aaaataaaaa aacgctagtt ttagtaactc gcgttgtttt cttcaccttt
3061 aataatagct actccaccac ttgttcctaa gcggtcagct cctgcttcaa tcattttttg
3121 agcatcttca aatgttctaa ctccaccagc tgctttaact aaagcattgt ctttaacaac
3181 tgacttcatt agtttaacat cttcaaatgt tgcacctgat tttgaaaatc ctgttgatgt
3241 tttaacaaat tctaatccag cttcaacagc tatttcacaa gctttcatga tttcttcttt
3301 tgttaataaa caattttcca taatacattt aacaacatgt gatccagctg ctttttttac
3361 agctttcatg tcttctaaaa ctaattcata atttttgtct tttaatgcac caatatttaa
3421 taccatatca atttctgttg caccatcttt aattgcttca gaaacttcga atgcttttgt
3481 agctgttgtg catgcaccta gaggaaaacc tacaacattt gttattccta catttgtgcc
3541 ttttaataat tctttacaat agcttgttca atatgaatta acacaaactg ttgcaaaatc
3601 aaattcaatt gcttcatcac ataattgttt aatttcagct ttcgtagcat cttgttttaa
3661 taatgtgtga tctatatatt tgtttagttt cattttttct cctatatatt catttttaat
3721 tttaattctt taataatttc gtctacttta actttagcgt tttgaacaga ttcaccaaca
3781 cctataaaat aaatttttag tttaggttca gttccacttg ggcgaacagc aaatcatgac
3841 ttatcttcta aataaaattt tagtaagtct tgtcctggca tattatacat tccatcgatg
3901 tagtcttcaa cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat
3961 gatttcatta atggttcaat ttttaatttc ttttcttctg gtttaaaatt caagtttaaa
4021 gtgaaagtgt aatatgcacc catttcttta aataaatctt ctaaatagtc tactaatgtt
4081 ttattttgtt ttttataaaa tcaagcagcc tctgctatta atatagaagc ttgtattcca
4141 tctttatctc tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca
4201 aaatttaatc cgttatcttc ttctttagca atttctctac ccattcattt aaatccagtt
4261 aaagttttta caatattaac tccatatttt tcatgagcga ttctatcacc caaatcactt
4321 gttacaaaac ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt
4381 gataattttc aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga
4441 ccatcatgtt ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata
4501 tctgcatcat gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga
4561 tttggattta caacattttt aaatgtttca tcttcaaatg catgctcttc aacctcaata
4621 acgttatatc ctgattcacg taatattttt ggggtaaatt tagttcctgt tccattaact
4681 gcgctaaaaa taatttttaa atctttttta gcttcttgct cttttttgta cgtctctgtt
4741 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
4801 accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc
4861 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
4921 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
4981 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
5041 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag
5101 cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg
5161 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
5221 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
5281 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
5341 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
5401 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
5461 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
5521 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
5581 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
5641 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
5701 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
5761 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
5821 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
5881 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
5941 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
6001 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
6061 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
6121 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
6181 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
6241 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
6301 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
6361 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
6421 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
6481 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
6541 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
6601 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
6661 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
6721 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
6781 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
6841 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
6901 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
6961 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
7021 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
7081 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
7141 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
7201 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
7261 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
7321 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
7381 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
7441 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
7501 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
7561 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
7621 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
7681 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
7741 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
7801 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
7861 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
7921 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
7981 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
8041 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
8101 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
8161 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
8221 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
8281 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
8341 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
8401 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
8461 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
8521 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
8581 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
8641 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
8701 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
8761 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
8821 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
8881 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
8941 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
9001 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
9061 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
9121 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
9181 gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc
9241 tggacaggct aagaagaaga aagattacaa agacgatgac gataagggat ccggcgcaac
9301 aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cgaccgagta
9361 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
9421 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
9481 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
9541 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
9601 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
9661 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
9721 cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
9781 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
9841 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
9901 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt
9961 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
10021 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
10081 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
10141 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
10201 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
10261 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
10321 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
10381 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
10441 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
10501 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact
10561 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
10621 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
10681 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
10741 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
10801 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagg
10861 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
10921 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
10981 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
11041 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
11101 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
11161 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
11221 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
11281 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
11341 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
11401 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
11461 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
11521 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
11581 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
11641 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
11701 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
11761 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
11821 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
11881 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
11941 ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat
12001 agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca
12061 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc
12121 ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg
12181 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg
12241 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact
12301 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt
12361 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac
12421 acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
12481 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
12541 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
12601 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
12661 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
12721 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
12781 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
12841 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
12901 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
12961 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
13021 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
13081 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
13141 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
13201 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
13261 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
13321 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
13381 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
13441 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
13501 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
13561 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
13621 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
13681 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
13741 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
13801 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
13861 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
13921 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
13981 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
14041 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
14101 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
14161 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
14221 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
14281 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
14341 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
14401 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
14461 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
14521 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
14581 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
14641 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
14701 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
14761 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
14821 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gac
//
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：pLentiCRISPR V2哺乳编辑质粒
别称：Plasmid#52961;LentiCRISPR V2
| 启动子: | U6 |
|---|---|
| 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： | 基因敲除 |
| 基因种属： | 空载体 |
| 基因类型： | CRISPR,Cas9,sgRNA |
| 原核抗性： | Amp |
| 真核抗性： | Puro |
| 荧光蛋白： |
质粒简介
plentiCRISPR V2哺乳编辑质粒
质粒图谱
质粒序列
LOCUS Exported 14873 bp ds-DNA circular SYN 24-JUL-2019
DEFINITION synthetic circular DNA
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14873)
AUTHORS Triple Threat
TITLE Direct Submission
FEATURES Location/Qualifiers
source 1..14873
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 238..617
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 618..821
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
LTR 835..1015
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/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 2440..2557
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2608..2848
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 2858..4737
/label=Stuffer
misc_RNA 4738..4813
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 4875..5086
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 5105..5114
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 5111..9214
/codon_start=1
/product="Cas9 (Csn1) endonuclease from the Streptococcus
pyogenes Type II CRISPR/Cas system"
/label=Cas9
/note="generates RNA-guided double strand breaks in DNA"
/translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLS-DAILL-SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLS-DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 9215..9262
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 9263..9286
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 9296..9352
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 9353..9949
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
misc_feature 9965..10553
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
LTR 10625..10858
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
polyA_signal 10890..11114
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 11160..11588
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 11602..11931
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 11782..11917
/label=SV40 ori
/note="SV40 origin of replication"
promoter 11979..12026
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 12045..12419
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 12549..12670
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(13121..13709)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(13880..14740)
/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"
promoter complement(14741..14845)
/gene="bla"
/label=AmpR promoter
BASE COUNT 3920 a 3699 c 3707 g 3546 t
ORIGIN
1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
2821 ttggctttat atatcttgtg gaaaggacga aacaccggag acggttgtaa atgagcacac
2881 aaaatacaca tgctaaaata ttatattcta tgacctttat aaaatcaacc aaaatcttct
2941 ttttaataac tttagtatca ataattagaa tttttatgtt cctttttgca aacttttaat
3001 aaaaatgagc aaaataaaaa aacgctagtt ttagtaactc gcgttgtttt cttcaccttt
3061 aataatagct actccaccac ttgttcctaa gcggtcagct cctgcttcaa tcattttttg
3121 agcatcttca aatgttctaa ctccaccagc tgctttaact aaagcattgt ctttaacaac
3181 tgacttcatt agtttaacat cttcaaatgt tgcacctgat tttgaaaatc ctgttgatgt
3241 tttaacaaat tctaatccag cttcaacagc tatttcacaa gctttcatga tttcttcttt
3301 tgttaataaa caattttcca taatacattt aacaacatgt gatccagctg ctttttttac
3361 agctttcatg tcttctaaaa ctaattcata atttttgtct tttaatgcac caatatttaa
3421 taccatatca atttctgttg caccatcttt aattgcttca gaaacttcga atgcttttgt
3481 agctgttgtg catgcaccta gaggaaaacc tacaacattt gttattccta catttgtgcc
3541 ttttaataat tctttacaat agcttgttca atatgaatta acacaaactg ttgcaaaatc
3601 aaattcaatt gcttcatcac ataattgttt aatttcagct ttcgtagcat cttgttttaa
3661 taatgtgtga tctatatatt tgtttagttt cattttttct cctatatatt catttttaat
3721 tttaattctt taataatttc gtctacttta actttagcgt tttgaacaga ttcaccaaca
3781 cctataaaat aaatttttag tttaggttca gttccacttg ggcgaacagc aaatcatgac
3841 ttatcttcta aataaaattt tagtaagtct tgtcctggca tattatacat tccatcgatg
3901 tagtcttcaa cattaacaac tttaagtcca gcaatttgag ttaagggtgt tgctctcaat
3961 gatttcatta atggttcaat ttttaatttc ttttcttctg gtttaaaatt caagtttaaa
4021 gtgaaagtgt aatatgcacc catttcttta aataaatctt ctaaatagtc tactaatgtt
4081 ttattttgtt ttttataaaa tcaagcagcc tctgctatta atatagaagc ttgtattcca
4141 tctttatctc tagctgagtc atcaattaca tatccataac tttcttcata agcaaaaaca
4201 aaatttaatc cgttatcttc ttctttagca atttctctac ccattcattt aaatccagtt
4261 aaagttttta caatattaac tccatatttt tcatgagcga ttctatcacc caaatcactt
4321 gttacaaaac ttgaatatag agccggattt tttggaatgc tatttaagcg ttttagattt
4381 gataattttc aatcaattaa aattggtcct gtttgatttc catctaatct tacaaaatga
4441 ccatcatgtt ttattgccat tccaaatctg tcagcatctg ggtcattcat aataataata
4501 tctgcatcat gtttaatacc atattcaagc ggtatttttc atgcaggatc aaattctgga
4561 tttggattta caacattttt aaatgtttca tcttcaaatg catgctcttc aacctcaata
4621 acgttatatc ctgattcacg taatattttt ggggtaaatt tagttcctgt tccattaact
4681 gcgctaaaaa taatttttaa atctttttta gcttcttgct cttttttgta cgtctctgtt
4741 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
4801 accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc
4861 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
4921 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
4981 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
5041 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag
5101 cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg
5161 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
5221 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
5281 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
5341 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
5401 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
5461 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
5521 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
5581 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
5641 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
5701 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
5761 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
5821 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
5881 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
5941 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
6001 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
6061 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
6121 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
6181 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
6241 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
6301 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
6361 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
6421 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
6481 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
6541 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
6601 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
6661 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
6721 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
6781 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
6841 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
6901 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
6961 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
7021 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
7081 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
7141 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
7201 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
7261 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
7321 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
7381 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
7441 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
7501 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
7561 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
7621 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
7681 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
7741 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
7801 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
7861 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
7921 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
7981 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
8041 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
8101 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
8161 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
8221 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
8281 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
8341 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
8401 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
8461 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
8521 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
8581 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
8641 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
8701 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
8761 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
8821 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
8881 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
8941 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
9001 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
9061 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
9121 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
9181 gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc
9241 tggacaggct aagaagaaga aagattacaa agacgatgac gataagggat ccggcgcaac
9301 aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cgaccgagta
9361 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
9421 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
9481 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
9541 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
9601 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
9661 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
9721 cctggccacc gtcggagtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
9781 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
9841 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
9901 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt
9961 cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt
10021 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc
10081 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga
10141 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc
10201 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct
10261 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg
10321 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct
10381 gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc
10441 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg
10501 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact
10561 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
10621 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
10681 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
10741 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
10801 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagg
10861 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
10921 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
10981 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
11041 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
11101 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
11161 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
11221 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
11281 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
11341 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
11401 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
11461 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
11521 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
11581 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
11641 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
11701 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
11761 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
11821 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
11881 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
11941 ggagcttgta tatccatttt cggatctgat cagcacgtgt tgacaattaa tcatcggcat
12001 agtatatcgg catagtataa tacgacaagg tgaggaacta aaccatggcc aagttgacca
12061 gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc tggaccgacc
12121 ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc cgggacgacg
12181 tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc ctggcctggg
12241 tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg tccacgaact
12301 tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg gggcgggagt
12361 tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag caggactgac
12421 acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
12481 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
12541 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
12601 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
12661 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
12721 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
12781 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
12841 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
12901 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
12961 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
13021 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
13081 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
13141 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
13201 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
13261 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
13321 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
13381 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
13441 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
13501 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
13561 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
13621 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
13681 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
13741 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
13801 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
13861 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
13921 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
13981 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
14041 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
14101 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
14161 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
14221 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
14281 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
14341 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
14401 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
14461 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
14521 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
14581 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
14641 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
14701 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
14761 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
14821 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gac
//
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
| P4151/pECMV-3×FLAG-UBE2U人源基因质粒 |
| P4152/pECMV-3×FLAG-UBTD1人源基因质粒 |
| P4153/pECMV-3×FLAG-VNN2(2同义突变）人源基因质粒 |
| P4154/pECMV-3×FLAG-LYN(1同义突变）人源基因质粒 |
| P4155/pECMV-3×FLAG-TOMM20人源基因质粒 |
| P4156/pECMV-3×FLAG-ZDHHC16人源基因质粒 |
| P4157/pECMV-3×FLAG-HMGB2人源基因质粒 |
| P4158/pECMV-3×FLAG-ATP6V1E2人源基因质粒 |
| P4159/pECMV-3×FLAG-CLEC4D人源基因质粒 |
| P4160/pECMV-3×FLAG-CDCP1人源基因质粒 |
| P4161/pECMV-3×FLAG-CARS人源基因质粒 |
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基因编辑再次升级！领域大牛刘如谦 Cell 发文开发新工具，可安全高效进行体内基因编辑
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GENETICS：用于检测哺乳动物细胞中 DNA-蛋白质交联质粒修复的简单、快速的定量分析
该方法的目的是提供灵活，快速和定量的技术来检测哺乳动物细胞系中DNA-蛋白质交联（DPC）修复的动力学。该测定不是全局测定异种生物诱导的或自发的染色体DPC去除的去除，而是检查在质粒DNA底物内的一个位点处特异性引入的均匀的，化学定义的损伤的修复。这种方法避免了放射性物质的使用，并且不依赖昂贵的或高度专业化的技术。相反，它依赖于标准的重组DNA程序和广泛可用的实时定量聚合酶链式反应（qPCR）仪器。考虑到所用策略的固有灵活性，可以改变交联蛋白的大小以及化学连接的性质和连接位点的精确 DNA
pLentiCRISPR V2哺乳编辑质粒
￥100 - 1000
