| 质粒名称: | pACT1:Cas9-GFP, U6:sgTK |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 122852 |
| 存储实验室: | David Sibley |
|
载体骨架基本信息 |
|
| 载体名称: | pUC19 |
| 载体出品公司: | New England Biolabs |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 2700 bp |
| 完整质粒大小: | 9981 bp |
| 载体类型: | CRISPR；Cas9-GFP质粒在隐孢子虫基因组编辑中的应用 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Cas9-GFP |
| 物种来源: | streptococcus pyogenes |
| 插入大小: | 4956 bp |
| 启动子 | Cryptosporidium parvum actin |
| 标签/融合蛋白: | eGFP（插入件上的C端子） |
| 克隆方法: | Gibson克隆 |
| 5' 测序引物 | CCCAACACTTAACCTTTCAGT |
| 3' 测序引物 | TCAGTTCTATTCTCATTTGAAAGCT |
|
文献引用方法 |
|
| 材料和方法部分: | pACT1:Cas9-GFP, U6:sgTK was a gift from David Sibley (Addgene plasmid # 122852 ; http://n2t.net/addgene:122852 ; RRID:Addgene_122852) |
| 参考文献部分: | A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability. Wilke G, Funkhouser-Jones LJ, Wang Y, Ravindran S, Wang Q, Beatty WL, Baldridge MT, VanDussen KL, Shen B, Kuhlenschmidt MS, Kuhlenschmidt TB, Witola WH, Stappenbeck TS, Sibley LD. Cell Host Microbe. 2019 Jun 18. pii: S1931-3128(19)30252-5. doi: 10.1016/j.chom.2019.05.007. 10.1016/j.chom.2019.05.007 PubMed 31231046 |
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pACT1:Cas9-GFP, U6:sgTK载体质粒基本信息
pACT1:Cas9-GFP, U6:sgTK质粒图谱载体图谱和pACT1:Cas9-GFP, U6:sgTK载体序列质粒序列多克隆位点信息
pACT1:Cas9-GFP, U6:sgTK质粒载体简介
pACT1:Cas9-GFP, U6:sgTK质粒序列载体序列
LOCUS pACT1:Cas9-GFP, U6:sgTK 9981 bp ds-DNA circular SYN 21-MAR-2019
DEFINITION Expresses Cas9 fused with GFP and a sgRNA targeting the
Cryptosporidium parvum TK gene.
ACCESSION .
VERSION .
KEYWORDS pACT1:Cas9-GFP, U6:sgTK
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9981)
TITLE Cas9-GFP plasmid for genome editing of Cryptosporidium parasites
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 9981)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Mar 21, 2019 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..9981
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 52..71
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 265..287
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 279..296
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 280..296
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
promoter 315..333
/label=SP6 promoter
/note="promoter for bacteriophage SP6 RNA polymerase"
primer_bind 315..332
/label=SP6
/note="SP6 promoter, forward primer"
misc_RNA complement(373..448)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS 2114..6217
/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
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 6221..6247
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 6266..6286
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6293..6313
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 6353..7069
/codon_start=1
/product="enhanced GFP with monomerizing A206K mutation
(Zacharias et al., 2002)"
/label=mEGFP
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
FVTAAGITLGMDELYK"
primer_bind complement(6395..6416)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind 7003..7024
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(7661..7677)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(7661..7677)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(7674..7696)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 7685..7701
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(7709..7739)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 7754..7775
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(7892..7909)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(8063..8651)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(8143..8162)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(8822..9682)
/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"
primer_bind 9445..9464
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(9683..9787)
/gene="bla"
/label=AmpR promoter
primer_bind 9855..9873
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
primer_bind complement(9911..9933)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
ORIGIN
1 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
61 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
121 gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat
181 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
241 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgaat
301 tcgagcgcca agctatttag gtgacactat agaatactca agctatgcat caagcttcct
361 aggaaaaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
421 tttaacttgc tatttctagc tctaaaacat tctagagcaa attgagccca acacttaacc
481 tttcagtttt tatattaaaa aaaactttat ttaacttcat aatgacgtgt ctgggtcttg
541 gcgcctaaat gttaataata aaccggtatt aagaaatggt ttgcataaaa tttgcatgca
601 atgacaggaa ttgagaattt ttgcatgtgg aagcggaata aatagtaaag gatagaattt
661 taagagtaga gtttgaataa aaaatattat tgattgataa taatacgaat taaatattaa
721 aaaaagagag tgaataaatg tttgaatact aaaaaaaata tataagtata aaatcaatta
781 atgctgcaat ataatgagca tataagagat atgggagaat actatagagg aatagttcca
841 atagtatatg gaagtaaaat attcccagga gaagagaaaa atcctgaaga agagtatttc
901 ttgactatat tgttgtgaga aatgtagggg agggagattt ggttaataat ttgcaagagt
961 tcattgtgtt gaatagaagc ttgtttgtct ttaaagaatg actgttgaag actagaattt
1021 ttccattctt catttctata taattggttg aagttggaaa gaataattga agactggttg
1081 gagaaatttg ggagctcggt acccggggat cctctagagt cgacctgcag tgggcttcag
1141 aatgagttgg ttataaacag taataataga aagcgttagg gctataaaaa tgaccaaata
1201 agtcatttag catcataagt atatacatta gatacagaat gagttcaaaa taaataaaaa
1261 attagactta cttacaaatt ctaagctata gtataaaaga actaagaaga tttgatgaat
1321 ttgctatacc attttaaata tttgtttata gttttttgat attaatataa ggactattaa
1381 gtgaaaagga aactacacat atttccgcca attcattgta ttgcatcgca cacataaatg
1441 aaagattaaa agaaaagaaa ttctattatc tagtattaaa ataataccaa taataagcaa
1501 cacttttttg ttatttttta ttataaagat taatttgcta ctttaaattg aaaaaaactt
1561 tacaggttta tacaaattcg tacatcacat gtatacaatg ctacatacat tacttgataa
1621 atattacgac gtgaaacgta ttaatttaaa cgaatcttag ttatgtaaat aaaaaaaggt
1681 tagattttag taaaaaaaaa agaccaaatt ttatattaaa tataaatctt gcattgaatg
1741 catttatgtc aaattgcatg caccttgccg cccaccatgc atagacgcgc gccaatacgt
1801 attaatagac gattacatga caaagtttag agattaaata aaaatcaagt ataaatttag
1861 aatagacaac atgtaaaaag tgagatgaat ctttttacga tacgtacagt gcgcattaaa
1921 aatccaaagt atatttttat tttcctgcgt gcgtatacat atctttaaat acttggataa
1981 agagataata agaattgtac ggcaagcaaa gtagtagttt aatagagcag tttttatatt
2041 ttatatatag cttacttaca atatagattt ttttttttca tagatttcgg gcatattaca
2101 caagtaacaa aagatggaca agaagtacag catcggcctg gacatcggca ccaactctgt
2161 gggctgggcc gtgatcaccg acgagtacaa ggtgcccagc aagaaattca aggtgctggg
2221 caacaccgac cggcacagca tcaagaagaa cctgatcggc gccctgctgt tcgacagcgg
2281 agaaacagcc gaggccaccc ggctgaagag aaccgccaga agaagataca ccagacggaa
2341 gaaccggatc tgctatctgc aagagatctt cagcaacgag atggccaagg tggacgacag
2401 cttcttccac agactggaag agtccttcct ggtggaagag gataagaagc acgagcggca
2461 ccccatcttc ggcaacatcg tggacgaggt ggcctaccac gagaagtacc ccaccatcta
2521 ccacctgaga aagaaactgg tggacagcac cgacaaggcc gacctgcggc tgatctatct
2581 ggccctggcc cacatgatca agttccgggg ccacttcctg atcgagggcg acctgaaccc
2641 cgacaacagc gacgtggaca agctgttcat ccagctggtg cagacctaca accagctgtt
2701 cgaggaaaac cccatcaacg ccagcggcgt ggacgccaag gccatcctgt ctgccagact
2761 gagcaagagc agacggctgg aaaatctgat cgcccagctg cccggcgaga agaagaatgg
2821 cctgttcggc aacctgattg ccctgagcct gggcctgacc cccaacttca agagcaactt
2881 cgacctggcc gaggatgcca aactgcagct gagcaaggac acctacgacg acgacctgga
2941 caacctgctg gcccagatcg gcgaccagta cgccgacctg tttctggccg ccaagaacct
3001 gtccgacgcc atcctgctga gcgacatcct gagagtgaac accgagatca ccaaggcccc
3061 cctgagcgcc tctatgatca agagatacga cgagcaccac caggacctga ccctgctgaa
3121 agctctcgtg cggcagcagc tgcctgagaa gtacaaagag attttcttcg accagagcaa
3181 gaacggctac gccggctaca tcgatggcgg agccagccag gaagagttct acaagttcat
3241 caagcccatc ctggaaaaga tggacggcac cgaggaactg ctcgtgaagc tgaacagaga
3301 ggacctgctg cggaagcagc ggaccttcga caacggcagc atcccccacc agatccacct
3361 gggagagctg cacgccattc tgcggcggca ggaagatttt tacccattcc tgaaggacaa
3421 ccgggaaaag atcgagaaga tcctgacctt ccgcatcccc tactacgtgg gccctctggc
3481 caggggaaac agcagattcg cctggatgac cagaaagagc gaggaaacca tcaccccctg
3541 gaacttcgag gaagtggtgg acaagggcgc cagcgcccag agcttcatcg agcggatgac
3601 caacttcgat aagaacctgc ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga
3661 gtacttcacc gtgtacaacg agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa
3721 gcccgccttc ctgagcggcg agcagaaaaa agccatcgtg gacctgctgt tcaagaccaa
3781 ccggaaagtg accgtgaagc agctgaaaga ggactacttc aagaaaatcg agtgcttcga
3841 ctccgtggaa atctccggcg tggaagatcg gttcaacgcc tccctgggca cataccacga
3901 tctgctgaaa attatcaagg acaaggactt cctggacaat gaggaaaacg aggacattct
3961 ggaagatatc gtgctgaccc tgacactgtt tgaggacaga gagatgatcg aggaacggct
4021 gaaaacctat gcccacctgt tcgacgacaa agtgatgaag cagctgaagc ggcggagata
4081 caccggctgg ggcaggctga gccggaagct gatcaacggc atccgggaca agcagtccgg
4141 caagacaatc ctggatttcc tgaagtccga cggcttcgcc aacagaaact tcatgcagct
4201 gatccacgac gacagcctga cctttaaaga ggacatccag aaagcccagg tgtccggcca
4261 gggcgatagc ctgcacgagc acattgccaa tctggccggc agccccgcca ttaagaaggg
4321 catcctgcag acagtgaagg tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc
4381 cgagaacatc gtgatcgaaa tggccagaga gaaccagacc acccagaagg gacagaagaa
4441 cagccgcgag agaatgaagc ggatcgaaga gggcatcaaa gagctgggca gccagatcct
4501 gaaagaacac cccgtggaaa acacccagct gcagaacgag aagctgtacc tgtactacct
4561 gcagaatggg cgggatatgt acgtggacca ggaactggac atcaaccggc tgtccgacta
4621 cgatgtggac catatcgtgc ctcagagctt tctgaaggac gactccatcg ataacaaagt
4681 gctgactcgg agcgacaaga accggggcaa gagcgacaac gtgccctccg aagaggtcgt
4741 gaagaagatg aagaactact ggcgccagct gctgaatgcc aagctgatta cccagaggaa
4801 gttcgacaat ctgaccaagg ccgagagagg cggcctgagc gaactggata aggccggctt
4861 catcaagaga cagctggtgg aaacccggca gatcacaaag cacgtggcac agatcctgga
4921 ctcccggatg aacactaagt acgacgagaa cgacaaactg atccgggaag tgaaagtgat
4981 caccctgaag tccaagctgg tgtccgattt ccggaaggat ttccagtttt acaaagtgcg
5041 cgagatcaac aactaccacc acgcccacga cgcctacctg aacgccgtcg tgggaaccgc
5101 cctgatcaaa aagtacccta agctggaaag cgagttcgtg tacggcgact acaaggtgta
5161 cgacgtgcgg aagatgatcg ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta
5221 cttcttctac agcaacatca tgaacttttt caagaccgag attaccctgg ccaacggcga
5281 gatccggaag cggcctctga tcgagacaaa cggcgaaaca ggcgagatcg tgtgggataa
5341 gggccgggac tttgccaccg tgcggaaagt gctgtctatg ccccaagtga atatcgtgaa
5401 aaagaccgag gtgcagacag gcggcttcag caaagagtct atcctgccca agaggaacag
5461 cgacaagctg atcgccagaa agaaggactg ggaccctaag aagtacggcg gcttcgacag
5521 ccccaccgtg gcctattctg tgctggtggt ggccaaagtg gaaaagggca agtccaagaa
5581 actgaagagt gtgaaagagc tgctggggat caccatcatg gaaagaagca gcttcgagaa
5641 gaatcccatc gactttctgg aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat
5701 caagctgcct aagtactccc tgttcgagct ggaaaacggc cggaagagaa tgctggcctc
5761 tgccggcgaa ctgcagaagg gaaacgaact ggccctgccc tccaaatatg tgaacttcct
5821 gtacctggcc agccactatg agaagctgaa gggctccccc gaggataatg agcagaaaca
5881 gctgtttgtg gaacagcaca aacactacct ggacgagatc atcgagcaga tcagcgagtt
5941 ctccaagaga gtgatcctgg ccgacgctaa tctggacaag gtgctgagcg cctacaacaa
6001 gcacagagac aagcctatca gagagcaggc cgagaatatc atccacctgt ttaccctgac
6061 caatctggga gcccctgccg ccttcaagta ctttgacacc accatcgacc ggaagaggta
6121 caccagcacc aaagaggtgc tggacgccac cctgatccac cagagcatca ccggcctgta
6181 cgagacacgg atcgacctgt ctcagctggg aggcgacgcc tatccctatg acgtgcccga
6241 ttatgccagc ctgggcagcg gctcccccaa gaaaaaacgc aaggtggaag atcctaagaa
6301 aaagcggaaa gtggacggca ttggtagtgg gagcaacggc agcagcggat ccgtgagcaa
6361 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
6421 cggccacaag ttcagcgtgc gcggcgaggg cgagggcgat gccaccaacg gcaagctgac
6481 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
6541 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
6601 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatctcct tcaaggacga
6661 cggcacctac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
6721 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
6781 caacttcaac agccacaacg tctatatcac ggccgacaag cagaagaacg gcatcaaggc
6841 gaacttcaag atccgccaca acgtcgagga cggcagcgtg cagctcgccg accactacca
6901 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
6961 ccagtccaag ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
7021 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaga tcaatattgt
7081 tcttggagtt gttcttaaca gcttatttcc tgaacacaga tacgtattgc aacttaaata
7141 gaaatatgaa ttacacaagt tatcacttct tgtagacaat ttcttattac attattcaga
7201 tctcagttct attctcattt gaaagctaaa aaaaaagatg ataaattgtc taatcaatgc
7261 taataaactc taaatttagc aagaaaattc tattataatt gttagtcaat gataactaat
7321 attatggata gaaggtgggc tagtttgaat caaaatagtt actaagtaat gtgtagttcg
7381 atacgatttt ttccttcttt ttaaacgaaa gtgtttaaat tatgtgcgtg ttgttttcct
7441 gagcacagta ttagaaatta attaagttga tttgtcaggt gatgtttatt accaaagttc
7501 aagtccgaaa atagagctgc catttaacaa taaaaaagag cggtccaaat ttaggcaaaa
7561 caaatttttt aaaaaaagct ccaagtccgg gaattgaacc cgggtcatcc gcgtgacagg
7621 cggatatact atgcaggcat gcaagcttgg cataatcatg gtcatagctg tttcctgtgt
7681 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
7741 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
7801 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
7861 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
7921 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
7981 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
8041 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
8101 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
8161 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
8221 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
8281 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
8341 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
8401 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
8461 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
8521 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
8581 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
8641 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
8701 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
8761 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
8821 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
8881 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
8941 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
9001 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
9061 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
9121 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
9181 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
9241 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
9301 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
9361 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
9421 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
9481 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
9541 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
9601 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
9661 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
9721 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
9781 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
9841 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
9901 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
9961 atgccgggag cagacaagcc c
//
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pACT1:Cas9-GFP, U6:sgTK质粒载体应用举例
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