| 质粒名称: | pCas9 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 162606 |
| 存储实验室: | Lars Steinmetz |
|
载体骨架基本信息 |
|
| 载体名称: | pRS415 |
| 载体出品公司: | Clontech |
| 空载体大小: | 4038 bp |
| 完整质粒大小: | 10247 bp |
| 载体类型: | Yeast Expression |
| 筛选标记: | LEU2 ; HygMX |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | Tef1-Cas9 |
| 物种来源: | Synthetic |
| 克隆方法: | Gibson Cloning |
|
文献引用方法 |
|
| 材料和方法部分: | pCas9 was a gift from Lars Steinmetz (Addgene plasmid # 162606 ; http://n2t.net/addgene:162606 ; RRID:Addgene_162606) |
| 参考文献部分: | CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and Bioinformatics Laboratory Module for Undergraduate and High School Students. Sankaran SM, Smith JD, Roy KR. J Microbiol Biol Educ. 2021 May 31;22(2):e00106-21. doi: 10.1128/jmbe.00106-21. eCollection 2021 Fall. 10.1128/jmbe.00106-21 PubMed 34594460 |
买家导航
pCas9载体质粒基本信息
pCas9质粒图谱载体图谱和pCas9载体序列质粒序列多克隆位点信息
pCas9质粒载体简介
pCas9质粒序列载体序列
LOCUS pCas9 12346 bp DNA circular SYN 14-APR-2025
DEFINITION Pre-express Cas9 in yeast.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 12346)
AUTHORS Sankaran SM, Smith JD, Roy KR
TITLE CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and
Bioinformatics Laboratory Module for Undergraduate and High School
Students.
JOURNAL J Microbiol Biol Educ. 2021 May 31;22(2):e00106-21. doi:
10.1128/jmbe.00106-21. eCollection 2021 Fall.
PUBMED 34594460
REFERENCE 2 (bases 1 to 12346)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Apr 14, 2025 from SnapGene Server 8.0.1
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1128/jmbe.00106-21"; journalName: "J Microbiol Biol Educ"; date:
"2021-05-31- 31"; volume: "22"; issue: "2"; pages: "e00106-21"
COMMENT Sequence Label: pCas9
FEATURES Location/Qualifiers
source 1..12346
/mol_type="other DNA"
/organism="synthetic DNA construct"
gene 1..1576
/label=hphMX6
/note="yeast selectable marker conferring hygromycin
resistance"
promoter 1..344
/label=TEF promoter
/note="Ashbya gossypii TEF promoter"
CDS 345..1373
/codon_start=1
/product="hygromycin B phosphotransferase"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
KE"
CDS 345..1373
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
KE"
terminator 1379..1576
/label=TEF terminator
/note="Ashbya gossypii TEF terminator"
promoter 1864..2271
/gene="S. cerevisiae LEU2"
/label=LEU2 promoter
CDS 2272..3366
/codon_start=1
/gene="S. cerevisiae LEU2"
/product="3-isopropylmalate dehydrogenase, required for
leucine biosynthesis"
/label=LEU2
/note="yeast auxotrophic marker"
/translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
DLGGSNSTTEVGDAVAEEVKKILA"
primer_bind complement(3860..3879)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 3979..4001
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4039..4057)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
misc_feature 4099..4602
/label=CEN/ARS
/note="S. cerevisiae CEN6 centromere fused to an
autonomously replicating sequence"
promoter 4639..4743
/gene="bla"
/label=AmpR promoter
CDS join(4744..4812,4813..5604)
/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 complement(4962..4981)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 5775..6363
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6264..6283
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 6517..6534
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 6651..6672
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter join(6687..6692,6693..6710,6711..6717)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 6725..6741
/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)."
primer_bind 6730..6752
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 6749..6765
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 6749..6765
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind 6784..6804
/label=T3
/note="T3 promoter, forward primer"
promoter 6786..6804
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
promoter 6822..7220
/gene="S. cerevisiae TEF1"
/label=TEF1 promoter
/note="promoter for EF-1-alpha"
primer_bind 7224..7240
/label=SK primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 7224..7240
/label=pBluescriptSK
/note="For pBluescript vector"
CDS 7252..11355
/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 7252..11355
/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 7252..11355
/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 11368..11388
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
terminator 11404..11651
/gene="S. cerevisiae CYC1"
/label=CYC1 terminator
/note="transcription terminator for CYC1"
primer_bind complement(11420..11438)
/label=CYC1
/note="CYC1 transcription termination signal, reverse
primer"
primer_bind complement(11669..11688)
/label=T7
/note="T7 promoter, forward primer"
promoter complement(11670..11688)
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
primer_bind complement(11698..11715)
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind complement(11698..11714)
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11707..11729)
/label=M13/pUC Forward
/note="In lacZ gene"
rep_origin 11855..12310
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11942..11961)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 12152..12173
/label=F1ori-F
/note="F1 origin, forward primer"
ORIGIN
1 gacatggagg cccagaatac cctccttgac agtcttgacg tgcgcagctc aggggcatga
61 tgtgactgtc gcccgtacat ttagcccata catccccatg tataatcatt tgcatccata
121 cattttgatg gccgcacggc gcgaagcaaa aattacggct cctcgctgcg gacctgcgag
181 cagggaaacg ctcccctcac agacgcgttg aattgtcccc acgccgcgcc cctgtagaga
241 aatataaaag gttaggattt gccactgagg ttcttctttc atatacttcc ttttaaaatc
301 ttgctaggat acagttctca catcacatcc gaacataaac aaccatgggt aaaaagcctg
361 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
421 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
481 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
541 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
601 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
661 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
721 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
781 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
841 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
901 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
961 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
1021 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
1081 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
1141 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
1201 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
1261 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
1321 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa taatcagtac
1381 tgacaataaa aagattcttg ttttcaagaa cttgtcattt gtatagtttt tttatattgt
1441 agttgttcta ttttaatcaa atgttagcgt gatttatatt ttttttcgcc tcgacatcat
1501 ctgcccagat gcgaagttaa gtgcgcagaa agtaatatca tgcgtcaatc gtatgtgaat
1561 gctggtcgct atactgctgt cgattcgata ctaacgccgc catccagtgt taattaacga
1621 cggtcgagga gaacttctag tatatctaca tacctaatat tattgcctta ttaaaaatgg
1681 aatcccaaca attacatcaa aatccacatt ctcttcaaaa tcaattgtcc tgtacttcct
1741 tgttcatgtg tgttcaaaaa cgttatattt ataggataat tatactctat ttctcaacaa
1801 gtaattggtt gtttggccga gcggtctaag gcgcctgatt caagaaatat cttgaccgca
1861 gttaactgtg ggaatactca ggtatcgtaa gatgcaagag ttcgaatctc ttagcaacca
1921 ttattttttt cctcaacata acgagaacac acaggggcgc tatcgcacag aatcaaattc
1981 gatgactgga aattttttgt taatttcaga ggtcgcctga cgcatatacc tttttcaact
2041 gaaaaattgg gagaaaaagg aaaggtgaga gcgccggaac cggcttttca tatagaatag
2101 agaagcgttc atgactaaat gcttgcatca caatacttga agttgacaat attatttaag
2161 gacctattgt tttttccaat aggtggttag caatcgtctt actttctaac ttttcttacc
2221 ttttacattt cagcaatata tatatatata tttcaaggat ataccattct aatgtctgcc
2281 cctaagaaga tcgtcgtttt gccaggtgac cacgttggtc aagaaatcac agccgaagcc
2341 attaaggttc ttaaagctat ttctgatgtt cgttccaatg tcaagttcga tttcgaaaat
2401 catttaattg gtggtgctgc tatcgatgct acaggtgttc cacttccaga tgaggcgctg
2461 gaagcctcca agaaggctga tgccgttttg ttaggtgctg tgggtggtcc taaatggggt
2521 accggtagtg ttagacctga acaaggttta ctaaaaatcc gtaaagaact tcaattgtac
2581 gccaacttaa gaccatgtaa ctttgcatcc gactctcttt tagacttatc tccaatcaag
2641 ccacaatttg ctaaaggtac tgacttcgtt gttgtcagag aattagtggg aggtatttac
2701 tttggtaaga gaaaggaaga cgatggtgat ggtgtcgctt gggatagtga acaatacacc
2761 gttccagaag tgcaaagaat cacaagaatg gccgctttca tggccctaca acatgagcca
2821 ccattgccta tttggtcctt ggataaagct aatgttttgg cctcttcaag attatggaga
2881 aaaactgtgg aggaaaccat caagaacgaa ttccctacat tgaaggttca acatcaattg
2941 attgattctg ccgccatgat cctagttaag aacccaaccc acctaaatgg tattataatc
3001 accagcaata tgtttggtga tatcatctcc gatgaagcct ccgttatccc aggttccttg
3061 ggtttgttgc catctgcgtc cttggcctct ttgccagaca agaacaccgc atttggtttg
3121 tacgaaccat gccacggttc tgctccagat ttgccaaaga ataaggtcaa ccctatcgcc
3181 actatcttgt ctgctgcaat gatgttgaaa ttgtcattga acttgcctga agaaggtaag
3241 gccattgaag atgcagttaa aaaggttttg gatgcaggta tcagaactgg tgatttaggt
3301 ggttccaaca gtaccaccga agtcggtgat gctgtcgccg aagaagttaa gaaaatcctt
3361 gcttaaaaag attctctttt tttatgatat ttgtacataa actttataaa tgaaattcat
3421 aatagaaacg acacgaaatt acaaaatgga atatgttcat agggtagacg aaactatata
3481 cgcaatctac atacatttat caagaaggag aaaaaggagg atgtaaagga atacaggtaa
3541 gcaaattgat actaatggct caacgtgata aggaaaaaga attgcacttt aacattaata
3601 ttgacaagga ggagggcacc acacaaaaag ttaggtgtaa cagaaaatca tgaaactatg
3661 attcctaatt tatatattgg aggattttct ctaaaaaaaa aaaaatacaa caaataaaaa
3721 acactcaatg acctgaccat ttgatggagt ttaagtcaat accttcttga accatttccc
3781 ataatggtga aagttccctc aagaatttta ctctgtcaga aacggcctta acgacgtagt
3841 cgatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
3901 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
3961 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
4021 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
4081 taatggtttc ttaggacgga tcgcttgcct gtaacttaca cgcgcctcgt atcttttaat
4141 gatggaataa tttgggaatt tactctgtgt ttatttattt ttatgttttg tatttggatt
4201 ttagaaagta aataaagaag gtagaagagt tacggaatga agaaaaaaaa ataaacaaag
4261 gtttaaaaaa tttcaacaaa aagcgtactt tacatatata tttattagac aagaaaagca
4321 gattaaatag atatacattc gattaacgat aagtaaaatg taaaatcaca ggattttcgt
4381 gtgtggtctt ctacacagac aagatgaaac aattcggcat taatacctga gagcaggaag
4441 aaaaagataa aaggtagtat ttgttggcga tccccctaga gtcttttaca tcttcggaaa
4501 acaaaaacta ttttttcttt aatttctttt tttactttct atttttaatt tatatattta
4561 tattaaaaaa tttaaattat aattattttt atagcacgtg atgaaaagga cccaggtggc
4621 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
4681 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
4741 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
4801 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
4861 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
4921 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
4981 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
5041 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
5101 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
5161 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
5221 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
5281 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
5341 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
5401 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
5461 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
5521 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
5581 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
5641 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
5701 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
5761 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
5821 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
5881 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
5941 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
6001 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
6061 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
6121 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
6181 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
6241 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
6301 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
6361 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
6421 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
6481 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
6541 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
6601 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
6661 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
6721 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
6781 cgcgcaatta accctcacta aagggaacaa aagctggagc tcatagcttc aaaatgtttc
6841 tactcctttt ttactcttcc agattttctc ggactccgcg catcgccgta ccacttcaaa
6901 acacccaagc acagcatact aaatttcccc tctttcttcc tctagggtgt cgttaattac
6961 ccgtactaaa ggtttggaaa agaaaaaaga gaccgcctcg tttctttttc ttcgtcgaaa
7021 aaggcaataa aaatttttat cacgtttctt tttcttgaaa attttttttt tgattttttt
7081 ctctttcgat gacctcccat tgatatttaa gttaataaac ggtcttcaat ttctcaagtt
7141 tcagtttcat ttttcttgtt ctattacaac tttttttact tcttgctcat tagaaagaaa
7201 gcatagcaat ctaatctaag ttttctagaa ctagtggatc ccccgggaaa aatggacaag
7261 aagtactcca ttgggctcga tatcggcaca aacagcgtcg gttgggccgt cattacggac
7321 gagtacaagg tgccgagcaa aaaattcaaa gttctgggca ataccgatcg ccacagcata
7381 aagaagaacc tcattggcgc cctcctgttc gactccgggg agacggccga agccacgcgg
7441 ctcaaaagaa cagcacggcg cagatatacc cgcagaaaga atcggatctg ctacctgcag
7501 gagatcttta gtaatgagat ggctaaggtg gatgactctt tcttccatag gctggaggag
7561 tcctttttgg tggaggagga taaaaagcac gagcgccacc caatctttgg caatatcgtg
7621 gacgaggtgg cgtaccatga aaagtaccca accatatatc atctgaggaa gaagcttgta
7681 gacagtactg ataaggctga cttgcggttg atctatctcg cgctggcgca tatgatcaaa
7741 tttcggggac acttcctcat cgagggggac ctgaacccag acaacagcga tgtcgacaaa
7801 ctctttatcc aactggttca gacttacaat cagcttttcg aagagaaccc gatcaacgca
7861 tccggagttg acgccaaagc aatcctgagc gctaggctgt ccaaatcccg gcggctcgaa
7921 aacctcatcg cacagctccc tggggagaag aagaacggcc tgtttggtaa tcttatcgcc
7981 ctgtcactcg ggctgacccc caactttaaa tctaacttcg acctggccga agatgccaag
8041 cttcaactga gcaaagacac ctacgatgat gatctcgaca atctgctggc ccagatcggc
8101 gaccagtacg cagacctttt tttggcggca aagaacctgt cagacgccat tctgctgagt
8161 gatattctgc gagtgaacac ggagatcacc aaagctccgc tgagcgctag tatgatcaag
8221 cgctatgatg agcaccacca agacttgact ttgctgaagg cccttgtcag acagcaactg
8281 cctgagaagt acaaggaaat tttcttcgat cagtctaaaa atggctacgc cggatacatt
8341 gacggcggag caagccagga ggaattttac aaatttatta agcccatctt ggaaaaaatg
8401 gacggcaccg aggagctgct ggtaaagctt aacagagaag atctgttgcg caaacagcgc
8461 actttcgaca atggaagcat cccccaccag attcacctgg gcgaactgca cgctatcctc
8521 aggcggcaag aggatttcta cccctttttg aaagataaca gggaaaagat tgagaaaatc
8581 ctcacatttc ggatacccta ctatgtaggc cccctcgccc ggggaaattc cagattcgcg
8641 tggatgactc gcaaatcaga agagaccatc actccctgga acttcgagga agtcgtggat
8701 aagggggcct ctgcccagtc cttcatcgaa aggatgacta actttgataa aaatctgcct
8761 aacgaaaagg tgcttcctaa acactctctg ctgtacgagt acttcacagt ttataacgag
8821 ctcaccaagg tcaaatacgt cacagaaggg atgagaaagc cagcattcct gtctggagag
8881 cagaagaaag ctatcgtgga cctcctcttc aagacgaacc ggaaagttac cgtgaaacag
8941 ctcaaagaag actatttcaa aaagattgaa tgtttcgact ctgttgaaat cagcggagtg
9001 gaggatcgct tcaacgcatc cctgggaacg tatcacgatc tcctgaaaat cattaaagac
9061 aaggacttcc tggacaatga ggagaacgag gacattcttg aggacattgt cctcaccctt
9121 acgttgtttg aagataggga gatgattgaa gaacgcttga aaacttacgc tcatctcttc
9181 gacgacaaag tcatgaaaca gctcaagagg cgccgatata caggatgggg gcggctgtca
9241 agaaaactga tcaatgggat ccgagacaag cagagtggaa agacaatcct ggattttctt
9301 aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga ctctctcacc
9361 tttaaggagg acatccagaa agcacaagtt tctggccagg gggacagtct tcacgagcac
9421 atcgctaatc ttgcaggtag cccagctatc aaaaagggaa tactgcagac cgttaaggtc
9481 gtggatgaac tcgtcaaagt aatgggaagg cataagcccg agaatatcgt tatcgagatg
9541 gcccgagaga accaaactac ccagaaggga cagaagaaca gtagggaaag gatgaagagg
9601 attgaagagg gtataaaaga actggggtcc caaatcctta aggaacaccc agttgaaaac
9661 acccagcttc agaatgagaa gctctacctg tactacctgc agaacggcag ggacatgtac
9721 gtggatcagg aactggacat caatcggctc tccgactacg acgtggatca tatcgtgccc
9781 cagtcttttc tcaaagatga ttctattgat aataaagtgt tgacaagatc cgataaaaat
9841 agagggaaga gtgataacgt cccctcagaa gaagttgtca agaaaatgaa aaattattgg
9901 cggcagctgc tgaacgccaa actgatcaca caacggaagt tcgataatct gactaaggct
9961 gaacgaggtg gcctgtctga gttggataaa gccggcttca tcaaaaggca gcttgttgag
10021 acacgccaga tcaccaagca cgtggcccaa attctcgatt cacgcatgaa caccaagtac
10081 gatgaaaatg acaaactgat tcgagaggtg aaagttatta ctctgaagtc taagctggtc
10141 tcagatttca gaaaggactt tcagttttat aaggtgagag agatcaacaa ttaccaccat
10201 gcgcatgatg cctacctgaa tgcagtggta ggcactgcac ttatcaaaaa atatcccaag
10261 cttgaatctg aatttgttta cggagactat aaagtgtacg atgttaggaa aatgatcgca
10321 aagtctgagc aggaaatagg caaggccacc gctaagtact tcttttacag caatattatg
10381 aattttttca agaccgagat tacactggcc aatggagaga ttcggaagcg accacttatc
10441 gaaacaaacg gagaaacagg agaaatcgtg tgggacaagg gtagggattt cgcgacagtc
10501 cggaaggtcc tgtccatgcc gcaggtgaac atcgttaaaa agaccgaagt acagaccgga
10561 ggcttctcca aggaaagtat cctcccgaaa aggaacagcg acaagctgat cgcacgcaaa
10621 aaagattggg accccaagaa atacggcgga ttcgattctc ctacagtcgc ttacagtgta
10681 ctggttgtgg ccaaagtgga gaaagggaag tctaaaaaac tcaaaagcgt caaggaactg
10741 ctgggcatca caatcatgga gcgatcaagc ttcgaaaaaa accccatcga ctttctcgag
10801 gcgaaaggat ataaagaggt caaaaaagac ctcatcatta agcttcccaa gtactctctc
10861 tttgagcttg aaaacggccg gaaacgaatg ctcgctagtg cgggcgagct gcagaaaggt
10921 aacgagctgg cactgccctc taaatacgtt aatttcttgt atctggccag ccactatgaa
10981 aagctcaaag ggtctcccga agataatgag cagaagcagc tgttcgtgga acaacacaaa
11041 cactaccttg atgagatcat cgagcaaata agcgaattct ccaaaagagt gatcctcgcc
11101 gacgctaacc tcgataaggt gctttctgct tacaataagc acagggataa gcccatcagg
11161 gagcaggcag aaaacattat ccacttgttt actctgacca acttgggcgc gcctgcagcc
11221 ttcaagtact tcgacaccac catagacaga aagcggtaca cctctacaaa ggaggtcctg
11281 gacgccacac tgattcatca gtcaattacg gggctctatg aaacaagaat cgacctctct
11341 cagctcggtg gagacagcag ggctgacccc aagaagaaga ggaaggtgtg atctcttctc
11401 gagtcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa
11461 ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta
11521 tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg
11581 tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg
11641 ctttaatttg cggccggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
11701 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
11761 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
11821 ttcccaacag ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc
11881 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
11941 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
12001 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
12061 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
12121 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
12181 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
12241 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
12301 cttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcgg
//
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pCas9质粒载体应用举例
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