| 质粒名称: | pAf-CRISPR-yA |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 191015 |
| 存储实验室: | Perng-Kuang Chang |
|
载体骨架基本信息 |
|
| 载体名称: | pPTRII |
| 载体出品公司: | TaKaRa |
| 空载体大小: | 10031bp |
| 完整质粒大小: | 13436bp |
| 载体类型: | CRISPR |
| 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: |
yA
gRNA/shRNA sequenceTTAGTGAGAATCATTTGGCG
|
| 物种来源: | Aspergillus flavus |
| 启动子: | Aspergillus flavus U6 |
| 标签/融合蛋白: | 人细胞色素c氧化酶亚基VIII（N端） |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | PstI (unknown if destroyed) |
| 3’克隆位点: | KpnI (unknown if destroyed) |
| 5’测序引物 | ATACTGCAGTTCTCTTTAGAATTCAACTGTGGGT |
| 3’测序引物 | TATGGTACCACATATTTAAAAAAAGTCTCCTGCC |
|
文献引用方法 |
|
| 材料和方法部分: | pAf-CRISPR-yA was a gift from Perng-Kuang Chang (Addgene plasmid # 191015 ; http://n2t.net/addgene:191015 ; RRID:Addgene_191015) |
| 参考文献部分: | A Simple CRISPR/Cas9 System for Efficiently Targeting Genes of Aspergillus Section Flavi Species, Aspergillus nidulans, Aspergillus fumigatus, Aspergillus terreus, and Aspergillus niger. Chang PK. Microbiol Spectr. 2023 Feb 14;11(1):e0464822. doi: 10.1128/spectrum.04648-22. Epub 2023 Jan 18. 10.1128/spectrum.04648-22 PubMed 36651760 |
买家导航
pAf-CRISPR-yA载体质粒基本信息
pAf-CRISPR-yA质粒图谱载体图谱和pAf-CRISPR-yA载体序列质粒序列多克隆位点信息
pAf-CRISPR-yA质粒载体简介
pAf-CRISPR-yA质粒序列载体序列
LOCUS pAf-CRISPR-yA 13436 bp DNA circular SYN 14-APR-2025
DEFINITION CRISPR vector based on Aspergillus flavus U6 promoter and terminator
for targeting the yA gene, which contains AMA1(the HindIII-PstI
fragment) and ptrA selection marker..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13436)
AUTHORS Chang PK
TITLE A Simple CRISPR/Cas9 System for Efficiently Targeting Genes of
Aspergillus Section Flavi Species, Aspergillus nidulans, Aspergillus
fumigatus, Aspergillus terreus, and Aspergillus niger.
JOURNAL Microbiol Spectr. 2023 Feb 14;11(1):e0464822. doi:
10.1128/spectrum.04648-22. Epub 2023 Jan 18.
PUBMED 36651760
REFERENCE 2 (bases 1 to 13436)
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"; journalName: "Microbiol
Spectr."; date: "2023-02-14"; volume: "11(1):e0464822. doi"; pages:
" 10.1128/spectrum.04648-22. Epub 2023 Jan 18"
COMMENT Sequence Label: pAf-CRISPR-yA
FEATURES Location/Qualifiers
source 1..13436
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(96..115)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 909..1497
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 1398..1417
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 1651..1668
/label=L4440
/note="L4440 vector, forward primer"
protein_bind 1785..1806
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
promoter join(1821..1826,1827..1844,1845..1851)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 1859..1875
/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 1864..1886
/label=M13/pUC Reverse
/note="In lacZ gene"
primer_bind 1883..1899
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind 1883..1899
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
intron 3020..3135
/gene="gpdA"
/label=gpdA intron
/note="intron from the Aspergillus nidulans
glyceraldehyde-3-phosphate dehydrogenase gene (Punt et al.,
1990)"
CDS 3156..3176
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 3207..7310
/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 3207..7310
/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 7323..7343
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
misc_RNA complement(7751..7826)
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
primer_bind 11149..11170
/label=F1ori-F
/note="F1 origin, forward primer"
gene complement(11289..13296)
/product="pyrithiamine resistance gene of Aspergillus
oryzae (Kubodera et al., 2000)"
/label=ptrA
/note="useful as a dominant selectable marker in
Aspergillus"
CDS complement(join(11533..12179,12238..12574))
/codon_start=1
/gene="ptrA of Aspergillus oryzae"
/product="pyrithiamine resistance protein (Kubodera et al.,
2000)"
/label=ptrA
/note="useful as a dominant selectable marker in
Aspergillus"
/translation="MSPPAAIYEPTVAATGLKGKVVVSETVPVEGASQTKLLDHFGGKW
DEFKFAPIRESQVSRAMTRRYFEDLDKYAESDVVIVGAGSCGLSTAYVLAKARPDLKIA
IVEASVSPGGGAWLGGQLFSAMVMRRPAEVFLNELGVPYEEDANPNYVVVKHASLFTST
LMSKVLSFPNVKLFNATAVEDLITRPTENGNPQIAGVVVNWTLVTLHHDDHSCMDPNTI
NAPVIISTTGHDGPFGAFCAKRLVSMGSVDKLGGMRGLDMSSAEDAIVKNTREVTKGLI
IGGMELSEIDGFNRMGPTFGAMVLSGVKAAEEALKVFDERQRECAE"
CDS 12972..12983
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
CDS join(13314..13382,13383..13436,1..738)
/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"
ORIGIN
1 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
61 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
121 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
181 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
241 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
301 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
361 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
421 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
481 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
541 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
601 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
661 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
721 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
781 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
841 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
901 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
961 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
1021 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
1081 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
1141 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
1201 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
1261 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
1321 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
1381 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
1441 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
1501 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
1561 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
1621 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
1681 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
1741 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
1801 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
1861 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcttcga
1921 gtaccattta attctatttg tgtttgatcg agacctaata cagcccctac aacgaccatc
1981 aaagtcgtat agctaccagt gaggaagtgg actcaaatcg acttcagcaa catctcctgg
2041 ataaacttta agcctaaact atacagaata agatggtgga gagcttatac cgagctccca
2101 aatctgtcca gatcatggtt gaccggtgcc tggatcttcc tatagaatca tccttattcg
2161 ttgacctagc tgattctgga gtgacccaga gggtcatgac ttgagcctaa aatccgccgc
2221 ctccaccatt tgtagaaaaa tgtgacgaac tcgtgagctc tgtacagtga ccggtgactc
2281 tttctggcat gcggagagac ggacggacgc agagagaagg gctgagtaat aagcgccact
2341 gcgccagaca gctctggcgg ctctgaggtg cagtggatga ttattaatcc gggaccggcc
2401 gcccctccgc cccgaagtgg aaaggctggt gtgcccctcg ttgaccaaga atctattgca
2461 tcatcggaga atatggagct tcatcgaatc accggcagta agcgaaggag aatgtgaagc
2521 caggggtgta tagccgtcgg cgaaatagca tgccattaac ctaggtacag aagtccaatt
2581 gcttccgatc tggtaaaaga ttcacgagat agtaccttct ccgaagtagg tagagcgagt
2641 acccggcgcg taagctccct aattggccca tccggcatct gtagggcgtc caaatatcgt
2701 gcctctcctg ctttgcccgg tgtatgaaac cggaaaggcc gctcaggagc tggccagcgg
2761 cgcagaccgg gaacacaagc tggcagtcga cccatccggt gctctgcact cgacctgctg
2821 aggtccctca gtccctggta ggcagctttg ccccgtctgt ccgcccggtg tgtcggcggg
2881 gttgacaagg tcgttgcgtc agtccaacat ttgttgccat attttcctgc tctccccacc
2941 agctgctctt ttcttttctc tttcttttcc catcttcagt atattcatct tcccatccaa
3001 gaacctttat ttcccctaag taagtacttt gctacatcca tactccatcc ttcccatccc
3061 ttattccttt gaacctttca gttcgagctt tcccacttca tcgcagcttg actaacagct
3121 accccgcttg agcaggcggc cgcatgatca tggccccaaa gaagaagcgg aaggtcggta
3181 tccacggagt cccagcagcc ggccgcatgg acaagaagta ctccatcggt ctcgacatcg
3241 gcaccaactc cgtcggttgg gccgtcatca ccgacgagta caaggtcccc tccaagaagt
3301 tcaaggtcct cggtaacacc gaccgtcact ccatcaagaa gaacctcatc ggtgccctcc
3361 tcttcgactc cggtgagacc gccgaggcca cccgtctcaa gcgtaccgcc cgtcgtcgtt
3421 acacccgtcg taagaaccgt atctgctacc tccaggagat cttctccaac gagatggcta
3481 aggtcgatga ctccttcttc caccgtctcg aggagtcctt cctcgtcgag gaggataaga
3541 agcacgagcg tcaccccatc ttcggtaaca tcgtcgacga ggtcgcctac cacgagaagt
3601 accccaccat ctaccacctc cgtaagaagc tggtcgactc tactgataag gctgaccttc
3661 gtctcatcta cctcgccctc gcccacatga tcaagttccg tggtcacttc ctcatcgagg
3721 gtgacctcaa ccctgacaac tccgatgtcg acaagctctt catccagctc gttcagactt
3781 acaaccagct cttcgaagag aaccccatca acgcttccgg tgttgacgcc aaggccatcc
3841 tctccgctcg tctctccaag tcccgtcgtc tcgagaacct catcgctcag ctccctggtg
3901 agaagaagaa cggtctcttc ggtaacctta tcgccctctc tctcggtctc acccccaact
3961 tcaagtccaa cttcgacctc gccgaggatg ccaaactcca gctctccaag gacacctacg
4021 atgacgatct cgacaacctc ctcgcccaga tcggtgacca gtacgctgac ctcttccttg
4081 ccgctaagaa cctctccgac gccatcctcc tctccgatat cctccgtgtc aacaccgaga
4141 tcaccaaggc tcccctctcc gcttctatga tcaagcgtta cgatgagcac caccaggacc
4201 tcactcttct caaggccctt gtccgtcagc agctccctga gaagtacaag gagatcttct
4261 tcgatcagtc caagaacggt tacgccggtt acatcgacgg cggtgcttcc caggaggagt
4321 tctacaagtt catcaagccc atcctcgaga agatggacgg caccgaggag ctcctcgtca
4381 agctcaaccg tgaggatctc ctccgtaagc agcgtacttt cgacaacggt tccatccccc
4441 accagatcca ccttggcgaa ctccacgcta tcctccgtcg tcaggaggac ttctacccct
4501 tcctcaagga caaccgtgag aagatcgaga agatcctcac cttccgtatc ccctactacg
4561 ttggccccct cgcccgtggt aactcccgtt tcgcctggat gactcgtaag tccgaagaga
4621 ccatcactcc ctggaacttc gaggaagtcg tcgataaggg tgcctctgcc cagtccttca
4681 tcgagcgtat gactaacttc gacaagaacc tccctaacga gaaggtcctt cctaagcact
4741 ctctccttta cgagtacttc actgtttaca acgagctcac caaggtcaag tacgtcaccg
4801 aaggtatgcg taagcccgcc ttcctctctg gtgagcagaa gaaggctatc gtcgacctcc
4861 tcttcaagac caaccgtaag gttaccgtca agcagctcaa ggaagactac ttcaagaaga
4921 tcgagtgctt cgactccgtt gagatctccg gtgtcgagga tcgtttcaac gcttccctcg
4981 gcacctacca cgatctcctc aagatcatca aggacaagga cttcctcgac aacgaggaga
5041 acgaggacat ccttgaggac atcgtcctca cccttaccct cttcgaggat cgtgagatga
5101 tcgaagaacg tctcaagact tacgctcacc tcttcgacga caaggtcatg aagcagctca
5161 agcgtcgtcg ttacactggt tggggccgtc tctcccgtaa gctgatcaac ggcatccgtg
5221 acaagcagtc tggtaagact atcctcgact tccttaagtc cgatggtttc gccaaccgta
5281 acttcatgca gctcatccac gacgactctc tcaccttcaa ggaggacatc cagaaggctc
5341 aggtttccgg tcagggtgac tcccttcacg agcacatcgc taacctcgcc ggttcccccg
5401 ctatcaagaa gggtatcctc cagaccgtta aggtcgtcga tgagctcgtc aaggttatgg
5461 gtcgtcacaa gcccgagaac atcgttatcg agatggcccg tgagaaccag actacccaga
5521 agggtcagaa gaactctcgt gaacgtatga agcgtatcga ggagggtatc aaggaactcg
5581 gctcccagat ccttaaggag caccccgtcg agaacaccca gcttcagaac gagaagctct
5641 acctctacta cctccagaac ggccgtgaca tgtacgttga tcaggagctc gacatcaacc
5701 gtctctccga ctacgacgtc gatcacatcg tcccccagtc cttcctcaag gatgactcta
5761 tcgacaacaa ggtcctcacc cgttccgata agaaccgtgg caagtctgac aacgtcccct
5821 ccgaagaggt tgtcaagaag atgaagaact actggcgtca gcttctcaac gccaagctca
5881 tcacccagcg taagttcgat aacctcacta aggctgaacg tggtggcctc tctgagctcg
5941 acaaggccgg cttcatcaag cgtcagcttg ttgagactcg tcagatcacc aagcacgtcg
6001 cccagattct cgactctcgc atgaacacca agtacgatga gaacgacaag ctcatccgtg
6061 aggtcaaggt tatcactctc aagtctaagc tcgtctccga cttccgtaag gacttccagt
6121 tctacaaggt ccgtgagatc aacaactacc accacgccca cgatgcctac ctcaacgctg
6181 tcgttggcac tgctcttatc aagaagtacc ccaagctgga gtccgagttc gtctacggtg
6241 actacaaggt ctacgatgtt cgtaagatga tcgctaagtc cgagcaggag atcggtaagg
6301 ccaccgctaa gtacttcttc tactccaaca tcatgaactt cttcaagacc gagatcaccc
6361 tcgccaacgg tgagatccgt aagcgtcccc tcatcgagac caacggtgag actggtgaga
6421 tcgtctggga caagggtcgt gacttcgcca ctgtccgtaa ggtcctctcc atgccccagg
6481 tcaacatcgt caagaagacc gaggtccaga ccggtggctt ctccaaggaa tctatcctcc
6541 ccaagcgtaa ctccgacaag ctcatcgctc gcaagaagga ctgggacccc aagaagtacg
6601 gcggtttcga ctctcccact gtcgcttact ccgtcctcgt tgtcgccaag gtcgagaagg
6661 gtaagtctaa gaagctcaag tccgtcaagg agctcctcgg catcaccatc atggagcgtt
6721 cctccttcga gaagaacccc atcgacttcc tcgaggccaa gggttacaag gaggtcaaga
6781 aggacctcat catcaagctg cccaagtact ccctcttcga gcttgagaac ggtcgtaagc
6841 gtatgctcgc ttccgccggt gagctccaga agggtaacga gctcgctctc ccctccaagt
6901 acgtcaactt cctctacctc gcctcccact acgagaagct caagggttct cccgaagaca
6961 acgagcagaa gcagctcttc gtcgagcagc acaagcacta ccttgatgag atcatcgagc
7021 agatctccga gttctccaag cgtgtcatcc tcgccgacgc taacctcgat aaggtcctct
7081 ccgcttacaa caagcaccgt gacaagccca tccgtgagca ggctgagaac atcatccacc
7141 tcttcactct caccaacctt ggtgcccctg ctgccttcaa gtacttcgac accaccatcg
7201 accgtaagcg ttacacctct accaaggagg tcctcgacgc cactctcatc caccagtcca
7261 ttaccggtct ctacgagact cgtatcgacc tctctcagct cggtggtgac tcccgtgctg
7321 accccaagaa gaagcgtaag gtctgagcta ggggaattcc ggaccggatc cacttaacgt
7381 tactgaaatc atcaaacagc ttgacgaatc tggatataag atcgttggtg tcgatgtcag
7441 ctccggagtt gagacaaatg gtgttcagga tctcgataag atacgttcat ttgtccaagc
7501 agcaaagagt gccttctagt gatttaatag ctccatgtca acaagaataa aacgcgtttc
7561 gggtttacct cttccagata cagctcatct gcaatgcatt aatgcattga ctgcaaccta
7621 gtaacggtac cacatattta aaaaaagtct cctgccctcc gtttcgaacc cgctccaata
7681 acaaaatagc cattctgcac ctccatacat tcctacctct atatcaagct gataaatgct
7741 caaaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt
7801 taacttgcta tttctagctc taaaacttag tgagaatcat ttggcgactt gttcttcttt
7861 acaatgattt atataccctt caaaagataa atctgagcag tctgattggc tgatggttta
7921 gttgagcggg aagcgtgggc ttacataagt gccaggaaca tcaaatgcga aaaccccgct
7981 ttgccgcctc tgaatcaccc ttagacgagg ttcgtattgt ttgtctatgt gctgagtttt
8041 ggagtagtct agaatacgat gagtaatact caatgcatgg agtccttact ttgtggtatg
8101 tatgtatctt ggctatcaat tgagtatctt tgtggctgag gagcacgagt tgggcgtaat
8161 ctgggtacaa gatatataac agactggttg ttaatcacat ttcaaaagaa aaagaaataa
8221 taaaaataga aaactgacta tgattgaatt agctggggat tgagaaagtc caggatttga
8281 tcaaagatgg agaagacaaa gagaatgaag caaaaagcaa aacccacagt tgaattctaa
8341 agagaactgc agattttgac ctagttaaca aggcacatca tgctatcact tccaacactc
8401 ttgctgagcg cgttctgcct tgcctttctg gttctacgaa gtatccagtc ccgcctgcaa
8461 cgccgcagaa acgccaaaag gtggggttgt caaccgccag ctagagctcc atcgggattc
8521 tttggcattc cgccgttcct cggtggtcgc agataactcc ctaaattccc tgagctaact
8581 ctaagtcgac catgccgttt atggttagcg cctcccaaaa aggaatggcc gacttagagt
8641 tacctcttga ccgacttttt ctttcctccc ccttacattt cgttaccaca acacattcct
8701 atatccaaac tccaggtaca taactagtcg aaatctcttt taaatctagt caagaactag
8761 atacttaacc ttcatactgc ttagagatag cctttgaagc tcttatttcg ctctttgtct
8821 cactctcacc ttcctcctcc tcctccaatc ctttttgctc gggactagtc caagactagt
8881 caacgattag tatgccgagc attcgcgata acgatctccg aaagtcccca gagtactgtc
8941 actatctcga ggcagttaag gacggggagc ttacgctgcc ggatttcaag atagtaagcc
9001 gacccgacta gctttaagcc tagttactga ctagtccaca ggacgacaat ggcgtgcctg
9061 atatccatcc atatgaagtc tactgccgag tgaagggatg cctcaagcgt acagtgagtc
9121 tactgctata ctagtttctg actagttatt aactagttga caggttccct ctgccaacag
9181 aaacatattg gtcaagcact tgaaggacaa gaactcccac ggcatggagt ttacattgca
9241 caatggacct cccactatga aggaactgat ggaggccaaa ggcaagtcct atctagatta
9301 cttggtgact agtccctgac tagtcaagta gcatggtatg aaggcttgtt tgaaggcact
9361 gttctcccaa ccccgactcc taccaagaag cgcaagcgag ctgcgtaagt ttctgtgagt
9421 ctaactagtg tattagctaa tatatagcag caccaagtcc aaggaccaca atactaaggg
9481 tgtcgagaat tcgtgagttt cttctcccat ttcaactagt ccttgactag tcactaacta
9541 ctttgcagca acgagggtga agctggaaat gatcaggaca atggcgaggg cccgtaagta
9601 cagccattca atgcagacta gttgctaact aatctgtgac tagttcaagt ggtccgtacg
9661 ccgtgcatac ccctgtgact ggtaggaatt tgagcaagcc tgtcttgccg cgcgatgaga
9721 aaggaaaggc aagttacatt cagccccgta cctaggatca gtgctaattt ataacctcta
9781 gccactcttt atgcagatcc gccgtgaggg tagcaaggca gctaaatcag ctggtgagaa
9841 aggaaccata ccctgcaaga cctgtcgcaa cgcaaagggc aaaggtaagc tatccaagct
9901 agtttgggac tagattctaa ctagtctcag caccgtgtgg ttcaaagcca tattgcgagt
9961 tttggcgctt tttctcatcg attgacgagg caaagggagc gagtatgcaa cctcgtaagt
10021 cagactcaga caaagaccaa ctagttattg accagtcttc tagaaggctc tgttgtggat
10081 cttgaggccc tggagagttc ctccaacaat ccggagacaa gcaagtcctc gtcggactag
10141 tcactaacta gactctaact agttgcagac atggataatg caaaagagac aagcaatgaa
10201 gaaagtggta agacatttct cctttgtggt tctggactag tctttgacta gtcacagtct
10261 taaacaagga aaatgagcat gaaaatgagg aggaaaaggc tgctgagccc gaggaagtgc
10321 agggtgatgg cagacatggt aggttaatac cttgttagtt attgctagtc actgactagt
10381 caataactag tctctgaaca ccttgcaatc actccgtttg cgcagctgaa cagtggtgag
10441 gataatagta gtaagttatt ctagcttcag agttatagga gactagatac taactagtat
10501 tagttgcaac taacctggat ctcagagact ttggcctcaa tctagaatct atctagttgt
10561 caactagact gtggtatcat tgtcttttat tttcctagtc ctggaactag cttctaacta
10621 gtctccctaa tatgtggctg tcttgttttt tttttttgtt tccctacccg gatatctagt
10681 ccccttctag gttctgttaa cctctcgggc tctgatttag tttaacgcaa acctgagatt
10741 agtttctaac tagtctctag gttttctatc cacctttaat tgtaataata aatacaagca
10801 acgtttatac gtcaaaagca tttataaact tttaccctaa agtagcttgc ttgtgtgttt
10861 agtttataat tagtctctta ttaatttgat gtaggtaagc ccgccacaaa tatatatttt
10921 taacaagata ccgtggaaaa acttcgtgct atcacaaaac agtatacaaa aaataagctg
10981 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
11041 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
11101 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
11161 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
11221 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
11281 ttaacaaaat ggggtgacga tgagccgctc ttgcatcttt gtttgtatta tactgtcttt
11341 cttgttacac ataattattc tagaatgccc caccgttaca tacgggacac agccatttac
11401 atatgcatgt ggattacgag ctaacgagtt cattcaaatc tcagaactat cacataatca
11461 tcattcccct atcgtcaaag accgtaagac aaatccggtt catgcactga acccattcgg
11521 gtagtgagtc atttactcag cacactcgcg ctgacgctcg tcgaacacct tcaatgcctc
11581 ctcggcagcc ttgacaccac tgagaaccat ggcaccgaag gtagggccca tgcggttaaa
11641 gccatcaatt tcagacagct ccataccgcc gattatcaag cccttagtaa cctcgcgggt
11701 gttcttgacg atggcatcct cggccgagct catgtcgaga ccacgcatgc cacctagctt
11761 gtcgacgctg cccatggaca ccaagcgctt cgcacagaag gcgccgaatg gcccatcgtg
11821 accagtggta ctgatgatga caggagcgtt gatagtgttg gggtccatgc aggagtgatc
11881 atcgtggtga agggtgacca gcgtccagtt gacgacaaca ccagcaatct gggggttgcc
11941 gttctcggtc ggacgggtga tcaagtcctc aacagcggta gcattgaaga gcttgacatt
12001 ggggaaggag agaaccttcg acatgagtgt cgaggtaaac agggaggcgt gcttgacgac
12061 aacgtagttg gggtttgcgt cctcttcgta aggaacaccc agctcgttca ggaagacttc
12121 cgcgggacgg cgcatgacca tagcagaaaa gagttggcca cccaaccagg caccgccacc
12181 tattgcacgt tagttccgga aagctgagtg caaggcaatc catcatggac tactgaccag
12241 gagagacgct ggcctcgacg atagcaatct tcaggtccgg acgagccttg gccaagacgt
12301 acgcagtgct cagaccgcag gaaccagcac caacaatgac aacgtcactt tcagcgtact
12361 tgtccaggtc ctcaaagtaa cgtctggtca tggcacgaga gacctggctt tcgcggatag
12421 gggcgaactt gaactcgtcc cacttgccac cgaaatggtc caacagcttg gtctgagaag
12481 ctccctcaac ggggacggtc tcagaaacca cgaccttacc cttgaggccg gtagcggcca
12541 cagtgggttc gtagatggca gctggaggag acatgtttca agttgcaatg actatcatct
12601 gttagccatt ccatcaacag gaagaacgag agaaggcatg acccttttcg ctggtattat
12661 ccagatcaag ttttagccgt ataatctcag aacgaaccca gtccatcgat gccatgtcct
12721 tctagactag gatcctagag tctagggccc agcttaggga gggcatgtga atgcatcgat
12781 gactgggaac gaacaccggc ccacgccaaa gacgttacct aagatacctt gatcattgtg
12841 agagtccagc caaaagtatt ccatgacttc catcgtatgc cctctagagg gctaatcgag
12901 gagtgtattt acattgtcgg ttggtttggg aactatagaa gatggtcagt tattccaatc
12961 accaaaggtt tatcgaaggg aggaagactt gttcagtttc gtccgaggac ttttggaatt
13021 caaatctgag atagagaatt gtgtgggatg agaggaaaag gaagaagatg gtggggttca
13081 gaaggagggg ttgagttaaa tagcatgggt tgagtcaacg tgataagggc actataccgt
13141 atagatcagc ggcacccgat tctatccgtt ccttttgctc ctctttagct ttgaccggtg
13201 agccggacaa gaaacaagtg aaatcatcct gacatcggcg gacgatctcc tagcttttac
13261 atttcgttac caatgggatc ccgtaatcaa ttgcccattg aaaaaggaag agtatgagta
13321 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
13381 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacga
//
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pAf-CRISPR-yA质粒载体应用举例
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