| 质粒名称: | pAf-CRISPR-phoA |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 207991 |
| 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: | 13733bp |
| 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: |
phoAgRNA/shRNA sequenceGTCATCCTTAGACGACACTC
|
| 物种来源: | Aspergillus flavus |
| 启动子: | Aspergillus flavus U6 |
| 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-phoA was a gift from Perng-Kuang Chang (Addgene plasmid # 207991 ; http://n2t.net/addgene:207991 ; RRID:Addgene_207991) |
| 参考文献部分: | Creating large chromosomal segment deletions in Aspergillus flavus by a dual CRISPR/Cas9 system: Deletion of gene clusters for production of aflatoxin, cyclopiazonic acid, and ustiloxin B. Chang PK. Fungal Genet Biol. 2023 Dec 27;170:103863. doi: 10.1016/j.fgb.2023.103863. 10.1016/j.fgb.2023.103863 PubMed 38154756 |
买家导航
pAf-CRISPR-phoA载体质粒基本信息
pAf-CRISPR-phoA质粒图谱载体图谱和pAf-CRISPR-phoA载体序列质粒序列多克隆位点信息
pAf-CRISPR-phoA质粒载体简介
pAf-CRISPR-phoA质粒序列载体序列
LOCUS pAf-CRISPR-phoA 13733 bp DNA circular SYN 25-AUG-2024
DEFINITION CRISPR vector used with pAf-CRISPR-ctfR1 (#207992) to delete both
aflatoxin and cyclopiazonic acid gene clusters of Aspergillus
flavus.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13733)
AUTHORS Chang PK
TITLE Creating large chromosomal segment deletions in Aspergillus flavus
by a dual CRISPR/Cas9 system: Deletion of gene clusters for
production of aflatoxin, cyclopiazonic acid, and ustiloxin B.
JOURNAL Fungal Genet Biol. 2023 Dec 27;170:103863. doi:
10.1016/j.fgb.2023.103863.
PUBMED 38154756
REFERENCE 2 (bases 1 to 13733)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 25, 2024 from SnapGene Server 7.0.3
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Fungal
Genet Biol."; date: "2023-12-27"; volume: "170:103863. doi"; pages:
" 10.1016/j.fgb.2023.103863"
FEATURES Location/Qualifiers
source 1..13733
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_RNA 3129..3204
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
CDS complement(3612..3632)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS complement(3645..7748)
/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 complement(3645..7748)
/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 complement(7779..7799)
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
intron 7820..7935
/gene="gpdA"
/label=gpdA intron
/note="intron from the Aspergillus nidulans
glyceraldehyde-3-phosphate dehydrogenase gene (Punt et al.,
1990)"
primer_bind complement(9056..9072)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(9056..9072)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(9069..9091)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 9080..9096
/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(9104..9134)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 9149..9170
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(9287..9304)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(9458..10046)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(9538..9557)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(10217..11077)
/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 10840..10859
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
gene 11095..13102
/product="pyrithiamine resistance gene of Aspergillus
oryzae (Kubodera et al., 2000)"
/label=ptrA
/note="useful as a dominant selectable marker in
Aspergillus"
CDS complement(11408..11419)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
CDS join(11817..12153,12212..12858)
/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"
primer_bind complement(13221..13242)
/label=F1ori-F
/note="F1 origin, forward primer"
ORIGIN
1 aaaaaaaaaa aacaagacag ccacatatta gggagactag ttagaagcta gttccaggac
61 taggaaaata aaagacaatg ataccacagt ctagttgaca actagataga ttctagattg
121 aggccaaagt ctctgagatc caggttagtt gcaactaata ctagttagta tctagtctcc
181 tataactctg aagctagaat aacttactac tattatcctc accactgttc agctgcgcaa
241 acggagtgat tgcaaggtgt tcagagacta gttattgact agtcagtgac tagcaataac
301 taacaaggta ttaacctacc atgtctgcca tcaccctgca cttcctcggg ctcagcagcc
361 ttttcctcct cattttcatg ctcattttcc ttgtttaaga ctgtgactag tcaaagacta
421 gtccagaacc acaaaggaga aatgtcttac cactttcttc attgcttgtc tcttttgcat
481 tatccatgtc tgcaactagt tagagtctag ttagtgacta gtccgacgag gacttgcttg
541 tctccggatt gttggaggaa ctctccaggg cctcaagatc cacaacagag ccttctagaa
601 gactggtcaa taactagttg gtctttgtct gagtctgact tacgaggttg catactcgct
661 ccctttgcct cgtcaatcga tgagaaaaag cgccaaaact cgcaatatgg ctttgaacca
721 cacggtgctg agactagtta gaatctagtc ccaaactagc ttggatagct tacctttgcc
781 ctttgcgttg cgacaggtct tgcagggtat ggttcctttc tcaccagctg atttagctgc
841 cttgctaccc tcacggcgga tctgcataaa gagtggctag aggttataaa ttagcactga
901 tcctaggtac ggggctgaat gtaacttgcc tttcctttct catcgcgcgg caagacaggc
961 ttgctcaaat tcctaccagt cacaggggta tgcacggcgt acggaccact tgaactagtc
1021 acagattagt tagcaactag tctgcattga atggctgtac ttacgggccc tcgccattgt
1081 cctgatcatt tccagcttca ccctcgttgc tgcaaagtag ttagtgacta gtcaaggact
1141 agttgaaatg ggagaagaaa ctcacgaatt ctcgacaccc ttagtattgt ggtccttgga
1201 cttggtgctg ctatatatta gctaatacac tagttagact cacagaaact tacgcagctc
1261 gcttgcgctt cttggtagga gtcggggttg ggagaacagt gccttcaaac aagccttcat
1321 accatgctac ttgactagtc agggactagt caccaagtaa tctagatagg acttgccttt
1381 ggcctccatc agttccttca tagtgggagg tccattgtgc aatgtaaact ccatgccgtg
1441 ggagttcttg tccttcaagt gcttgaccaa tatgtttctg ttggcagagg gaacctgtca
1501 actagttaat aactagtcag aaactagtat agcagtagac tcactgtacg cttgaggcat
1561 cccttcactc ggcagtagac ttcatatgga tggatatcag gcacgccatt gtcgtcctgt
1621 ggactagtca gtaactaggc ttaaagctag tcgggtcggc ttactatctt gaaatccggc
1681 agcgtaagct ccccgtcctt aactgcctcg agatagtgac agtactctgg ggactttcgg
1741 agatcgttat cgcgaatgct cggcatacta atcgttgact agtcttggac tagtcccgag
1801 caaaaaggat tggaggagga ggaggaaggt gagagtgaga caaagagcga aataagagct
1861 tcaaaggcta tctctaagca gtatgaaggt taagtatcta gttcttgact agatttaaaa
1921 gagatttcga ctagttatgt acctggagtt tggatatagg aatgtgttgt ggtaacgaaa
1981 tgtaaggggg aggaaagaaa aagtcggtca agaggtaact ctaagtcggc cattcctttt
2041 tgggaggcgc taaccataaa cggcatggtc gacttagagt tagctcaggg aatttaggga
2101 gttatctgcg accaccgagg aacggcggaa tgccaaagaa tcccgatgga gctctagctg
2161 gcggttgaca accccacctt ttggcgtttc tgcggcgttg caggcgggac tggatacttc
2221 gtagaaccag aaaggcaagg cagaacgcgc tcagcaagag tgttggaagt gatagcatga
2281 tgtgccttgt taactaggtc aaaatctgca atatgcttga tgttatccaa agtgtgagag
2341 aggaaggtcc aaacatacac gattgggaga gggcctaggt ataagagttt ttgagtagaa
2401 cgcatgtgag cccagccatc tcgaggagat taaacacggg ccggcatttg atggctatgt
2461 tagtacccca atggaaagcc tgagagtcca gtggtcgcag ataactccct aaattccctg
2521 agctaactct aagtcgacca tgccgtttat ggttagcgcc tcccaaaaag gaatggccga
2581 cttagagtta cctcttgacc gactgcagtt ctctttagaa ttcaactgtg ggttttgctt
2641 tttgcttcat tctctttgtc ttctccatct ttgatcaaat cctggacttt ctcaatcccc
2701 agctaattca atcatagtca gttttctatt tttattattt ctttttcttt tgaaatgtga
2761 ttaacaacca gtctgttata tatcttgtac ccagattacg cccaactcgt gctcctcagc
2821 cacaaagata ctcaattgat agccaagata catacatacc acaaagtaag gactccatgc
2881 attgagtatt actcatcgta ttctagacta ctccaaaact cagcacatag acaaacaata
2941 cgaacctcgt ctaagggtga ttcagaggcg gcaaagcggg gttttcgcat ttgatgttcc
3001 tggcacttat gtaagcccac gcttcccgct caactaaacc atcagccaat cagactgctc
3061 agatttatct tttgaagggt atataaatca ttgtaaagaa gaacaagtgt catccttaga
3121 cgacactcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
3181 gaaaaagtgg caccgagtcg gtgctttttt tttgagcatt tatcagcttg atatagaggt
3241 aggaatgtat ggaggtgcag aatggctatt ttgttattgg agcgggttcg aaacggaggg
3301 caggagactt tttttaaata tgtggtaccg ttactaggtt gcagtcaatg cattaatgca
3361 ttgcagatga gctgtatctg gaagaggtaa acccgaaacg cgttttattc ttgttgacat
3421 ggagctatta aatcactaga aggcactctt tgctgcttgg acaaatgaac gtatcttatc
3481 gagatcctga acaccatttg tctcaactcc ggagctgaca tcgacaccaa cgatcttata
3541 tccagattcg tcaagctgtt tgatgatttc agtaacgtta agtggatccg gtccggaatt
3601 cccctagctc agaccttacg cttcttcttg gggtcagcac gggagtcacc accgagctga
3661 gagaggtcga tacgagtctc gtagagaccg gtaatggact ggtggatgag agtggcgtcg
3721 aggacctcct tggtagaggt gtaacgctta cggtcgatgg tggtgtcgaa gtacttgaag
3781 gcagcagggg caccaaggtt ggtgagagtg aagaggtgga tgatgttctc agcctgctca
3841 cggatgggct tgtcacggtg cttgttgtaa gcggagagga ccttatcgag gttagcgtcg
3901 gcgaggatga cacgcttgga gaactcggag atctgctcga tgatctcatc aaggtagtgc
3961 ttgtgctgct cgacgaagag ctgcttctgc tcgttgtctt cgggagaacc cttgagcttc
4021 tcgtagtggg aggcgaggta gaggaagttg acgtacttgg aggggagagc gagctcgtta
4081 cccttctgga gctcaccggc ggaagcgagc atacgcttac gaccgttctc aagctcgaag
4141 agggagtact tgggcagctt gatgatgagg tccttcttga cctccttgta acccttggcc
4201 tcgaggaagt cgatggggtt cttctcgaag gaggaacgct ccatgatggt gatgccgagg
4261 agctccttga cggacttgag cttcttagac ttacccttct cgaccttggc gacaacgagg
4321 acggagtaag cgacagtggg agagtcgaaa ccgccgtact tcttggggtc ccagtccttc
4381 ttgcgagcga tgagcttgtc ggagttacgc ttggggagga tagattcctt ggagaagcca
4441 ccggtctgga cctcggtctt cttgacgatg ttgacctggg gcatggagag gaccttacgg
4501 acagtggcga agtcacgacc cttgtcccag acgatctcac cagtctcacc gttggtctcg
4561 atgaggggac gcttacggat ctcaccgttg gcgagggtga tctcggtctt gaagaagttc
4621 atgatgttgg agtagaagaa gtacttagcg gtggccttac cgatctcctg ctcggactta
4681 gcgatcatct tacgaacatc gtagaccttg tagtcaccgt agacgaactc ggactccagc
4741 ttggggtact tcttgataag agcagtgcca acgacagcgt tgaggtaggc atcgtgggcg
4801 tggtggtagt tgttgatctc acggaccttg tagaactgga agtccttacg gaagtcggag
4861 acgagcttag acttgagagt gataaccttg acctcacgga tgagcttgtc gttctcatcg
4921 tacttggtgt tcatgcgaga gtcgagaatc tgggcgacgt gcttggtgat ctgacgagtc
4981 tcaacaagct gacgcttgat gaagccggcc ttgtcgagct cagagaggcc accacgttca
5041 gccttagtga ggttatcgaa cttacgctgg gtgatgagct tggcgttgag aagctgacgc
5101 cagtagttct tcatcttctt gacaacctct tcggagggga cgttgtcaga cttgccacgg
5161 ttcttatcgg aacgggtgag gaccttgttg tcgatagagt catccttgag gaaggactgg
5221 gggacgatgt gatcgacgtc gtagtcggag agacggttga tgtcgagctc ctgatcaacg
5281 tacatgtcac ggccgttctg gaggtagtag aggtagagct tctcgttctg aagctgggtg
5341 ttctcgacgg ggtgctcctt aaggatctgg gagccgagtt ccttgatacc ctcctcgata
5401 cgcttcatac gttcacgaga gttcttctga cccttctggg tagtctggtt ctcacgggcc
5461 atctcgataa cgatgttctc gggcttgtga cgacccataa ccttgacgag ctcatcgacg
5521 accttaacgg tctggaggat acccttcttg atagcggggg aaccggcgag gttagcgatg
5581 tgctcgtgaa gggagtcacc ctgaccggaa acctgagcct tctggatgtc ctccttgaag
5641 gtgagagagt cgtcgtggat gagctgcatg aagttacggt tggcgaaacc atcggactta
5701 aggaagtcga ggatagtctt accagactgc ttgtcacgga tgccgttgat cagcttacgg
5761 gagagacggc cccaaccagt gtaacgacga cgcttgagct gcttcatgac cttgtcgtcg
5821 aagaggtgag cgtaagtctt gagacgttct tcgatcatct cacgatcctc gaagagggta
5881 agggtgagga cgatgtcctc aaggatgtcc tcgttctcct cgttgtcgag gaagtccttg
5941 tccttgatga tcttgaggag atcgtggtag gtgccgaggg aagcgttgaa acgatcctcg
6001 acaccggaga tctcaacgga gtcgaagcac tcgatcttct tgaagtagtc ttccttgagc
6061 tgcttgacgg taaccttacg gttggtcttg aagaggaggt cgacgatagc cttcttctgc
6121 tcaccagaga ggaaggcggg cttacgcata ccttcggtga cgtacttgac cttggtgagc
6181 tcgttgtaaa cagtgaagta ctcgtaaagg agagagtgct taggaaggac cttctcgtta
6241 gggaggttct tgtcgaagtt agtcatacgc tcgatgaagg actgggcaga ggcaccctta
6301 tcgacgactt cctcgaagtt ccagggagtg atggtctctt cggacttacg agtcatccag
6361 gcgaaacggg agttaccacg ggcgaggggg ccaacgtagt aggggatacg gaaggtgagg
6421 atcttctcga tcttctcacg gttgtccttg aggaaggggt agaagtcctc ctgacgacgg
6481 aggatagcgt ggagttcgcc aaggtggatc tggtggggga tggaaccgtt gtcgaaagta
6541 cgctgcttac ggaggagatc ctcacggttg agcttgacga ggagctcctc ggtgccgtcc
6601 atcttctcga ggatgggctt gatgaacttg tagaactcct cctgggaagc accgccgtcg
6661 atgtaaccgg cgtaaccgtt cttggactga tcgaagaaga tctccttgta cttctcaggg
6721 agctgctgac ggacaagggc cttgagaaga gtgaggtcct ggtggtgctc atcgtaacgc
6781 ttgatcatag aagcggagag gggagccttg gtgatctcgg tgttgacacg gaggatatcg
6841 gagaggagga tggcgtcgga gaggttctta gcggcaagga agaggtcagc gtactggtca
6901 ccgatctggg cgaggaggtt gtcgagatcg tcatcgtagg tgtccttgga gagctggagt
6961 ttggcatcct cggcgaggtc gaagttggac ttgaagttgg gggtgagacc gagagagagg
7021 gcgataaggt taccgaagag accgttcttc ttctcaccag ggagctgagc gatgaggttc
7081 tcgagacgac gggacttgga gagacgagcg gagaggatgg ccttggcgtc aacaccggaa
7141 gcgttgatgg ggttctcttc gaagagctgg ttgtaagtct gaacgagctg gatgaagagc
7201 ttgtcgacat cggagttgtc agggttgagg tcaccctcga tgaggaagtg accacggaac
7261 ttgatcatgt gggcgagggc gaggtagatg agacgaaggt cagccttatc agtagagtcg
7321 accagcttct tacggaggtg gtagatggtg gggtacttct cgtggtaggc gacctcgtcg
7381 acgatgttac cgaagatggg gtgacgctcg tgcttcttat cctcctcgac gaggaaggac
7441 tcctcgagac ggtggaagaa ggagtcatcg accttagcca tctcgttgga gaagatctcc
7501 tggaggtagc agatacggtt cttacgacgg gtgtaacgac gacgggcggt acgcttgaga
7561 cgggtggcct cggcggtctc accggagtcg aagaggaggg caccgatgag gttcttcttg
7621 atggagtgac ggtcggtgtt accgaggacc ttgaacttct tggaggggac cttgtactcg
7681 tcggtgatga cggcccaacc gacggagttg gtgccgatgt cgagaccgat ggagtacttc
7741 ttgtccatgc ggccggctgc tgggactccg tggataccga ccttccgctt cttctttggg
7801 gccatgatca tgcggccgcc tgctcaagcg gggtagctgt tagtcaagct gcgatgaagt
7861 gggaaagctc gaactgaaag gttcaaagga ataagggatg ggaaggatgg agtatggatg
7921 tagcaaagta cttacttagg ggaaataaag gttcttggat gggaagatga atatactgaa
7981 gatgggaaaa gaaagagaaa agaaaagagc agctggtggg gagagcagga aaatatggca
8041 acaaatgttg gactgacgca acgaccttgt caaccccgcc gacacaccgg gcggacagac
8101 ggggcaaagc tgcctaccag ggactgaggg acctcagcag gtcgagtgca gagcaccgga
8161 tgggtcgact gccagcttgt gttcccggtc tgcgccgctg gccagctcct gagcggcctt
8221 tccggtttca tacaccgggc aaagcaggag aggcacgata tttggacgcc ctacagatgc
8281 cggatgggcc aattagggag cttacgcgcc gggtactcgc tctacctact tcggagaagg
8341 tactatctcg tgaatctttt accagatcgg aagcaattgg acttctgtac ctaggttaat
8401 ggcatgctat ttcgccgacg gctatacacc cctggcttca cattctcctt cgcttactgc
8461 cggtgattcg atgaagctcc atattctccg atgatgcaat agattcttgg tcaacgaggg
8521 gcacaccagc ctttccactt cggggcggag gggcggccgg tcccggatta ataatcatcc
8581 actgcacctc agagccgcca gagctgtctg gcgcagtggc gcttattact cagcccttct
8641 ctctgcgtcc gtccgtctct ccgcatgcca gaaagagtca ccggtcactg tacagagctc
8701 acgagttcgt cacatttttc tacaaatggt ggaggcggcg gattttaggc tcaagtcatg
8761 accctctggg tcactccaga atcagctagg tcaacgaata aggatgattc tataggaaga
8821 tccaggcacc ggtcaaccat gatctggaca gatttgggag ctcggtataa gctctccacc
8881 atcttattct gtatagttta ggcttaaagt ttatccagga gatgttgctg aagtcgattt
8941 gagtccactt cctcactggt agctatacga ctttgatggt cgttgtaggg gctgtattag
9001 gtctcgatca aacacaaata gaattaaatg gtactcgaag cttggcgtaa tcatggtcat
9061 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
9121 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
9181 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
9241 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
9301 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
9361 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
9421 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
9481 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
9541 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
9601 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
9661 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
9721 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
9781 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
9841 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
9901 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
9961 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
10021 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
10081 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
10141 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
10201 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
10261 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
10321 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
10381 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
10441 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
10501 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
10561 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
10621 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
10681 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
10741 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
10801 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
10861 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
10921 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
10981 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
11041 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatgggcaa
11101 ttgattacgg gatcccattg gtaacgaaat gtaaaagcta ggagatcgtc cgccgatgtc
11161 aggatgattt cacttgtttc ttgtccggct caccggtcaa agctaaagag gagcaaaagg
11221 aacggataga atcgggtgcc gctgatctat acggtatagt gcccttatca cgttgactca
11281 acccatgcta tttaactcaa cccctccttc tgaaccccac catcttcttc cttttcctct
11341 catcccacac aattctctat ctcagatttg aattccaaaa gtcctcggac gaaactgaac
11401 aagtcttcct cccttcgata aacctttggt gattggaata actgaccatc ttctatagtt
11461 cccaaaccaa ccgacaatgt aaatacactc ctcgattagc cctctagagg gcatacgatg
11521 gaagtcatgg aatacttttg gctggactct cacaatgatc aaggtatctt aggtaacgtc
11581 tttggcgtgg gccggtgttc gttcccagtc atcgatgcat tcacatgccc tccctaagct
11641 gggccctaga ctctaggatc ctagtctaga aggacatggc atcgatggac tgggttcgtt
11701 ctgagattat acggctaaaa cttgatctgg ataataccag cgaaaagggt catgccttct
11761 ctcgttcttc ctgttgatgg aatggctaac agatgatagt cattgcaact tgaaacatgt
11821 ctcctccagc tgccatctac gaacccactg tggccgctac cggcctcaag ggtaaggtcg
11881 tggtttctga gaccgtcccc gttgagggag cttctcagac caagctgttg gaccatttcg
11941 gtggcaagtg ggacgagttc aagttcgccc ctatccgcga aagccaggtc tctcgtgcca
12001 tgaccagacg ttactttgag gacctggaca agtacgctga aagtgacgtt gtcattgttg
12061 gtgctggttc ctgcggtctg agcactgcgt acgtcttggc caaggctcgt ccggacctga
12121 agattgctat cgtcgaggcc agcgtctctc ctggtcagta gtccatgatg gattgccttg
12181 cactcagctt tccggaacta acgtgcaata ggtggcggtg cctggttggg tggccaactc
12241 ttttctgcta tggtcatgcg ccgtcccgcg gaagtcttcc tgaacgagct gggtgttcct
12301 tacgaagagg acgcaaaccc caactacgtt gtcgtcaagc acgcctccct gtttacctcg
12361 acactcatgt cgaaggttct ctccttcccc aatgtcaagc tcttcaatgc taccgctgtt
12421 gaggacttga tcacccgtcc gaccgagaac ggcaaccccc agattgctgg tgttgtcgtc
12481 aactggacgc tggtcaccct tcaccacgat gatcactcct gcatggaccc caacactatc
12541 aacgctcctg tcatcatcag taccactggt cacgatgggc cattcggcgc cttctgtgcg
12601 aagcgcttgg tgtccatggg cagcgtcgac aagctaggtg gcatgcgtgg tctcgacatg
12661 agctcggccg aggatgccat cgtcaagaac acccgcgagg ttactaaggg cttgataatc
12721 ggcggtatgg agctgtctga aattgatggc tttaaccgca tgggccctac cttcggtgcc
12781 atggttctca gtggtgtcaa ggctgccgag gaggcattga aggtgttcga cgagcgtcag
12841 cgcgagtgtg ctgagtaaat gactcactac ccgaatgggt tcagtgcatg aaccggattt
12901 gtcttacggt ctttgacgat aggggaatga tgattatgtg atagttctga gatttgaatg
12961 aactcgttag ctcgtaatcc acatgcatat gtaaatggct gtgtcccgta tgtaacggtg
13021 gggcattcta gaataattat gtgtaacaag aaagacagta taatacaaac aaagatgcaa
13081 gagcggctca tcgtcacccc attttgttaa aattcgcgtt aaatttttgt taaatcagct
13141 cattttttaa ccaatagacc gaaatcggca aaatccctta taaatcaaaa gaatagcccg
13201 agatagagtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
13261 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
13321 ccaaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
13381 gcccccgatt tagagcttga cggggaaagc cagcttattt tttgtatact gttttgtgat
13441 agcacgaagt ttttccacgg tatcttgtta aaaatatata tttgtggcgg gcttacctac
13501 atcaaattaa taagagacta attataaact aaacacacaa gcaagctact ttagggtaaa
13561 agtttataaa tgcttttgac gtataaacgt tgcttgtatt tattattaca attaaaggtg
13621 gatagaaaac ctagagacta gttagaaact aatctcaggt ttgcgttaaa ctaaatcaga
13681 gcccgagagg ttaacagaac ctagaagggg actagatatc cgggtaggga aac
//
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pAf-CRISPR-phoA质粒载体应用举例
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