| 质粒名称: | pET28a/Cas9-Cys |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 53261 |
| 存储实验室: | Hyongbum Kim |
|
载体骨架基本信息 |
|
| 载体名称: | pET28a |
| 载体出品公司: | Novagen |
| 载体抗性: | 卡那霉素 |
| 空载体大小: | 5369 bp |
| 完整质粒大小: | 9542 bp |
| 载体修饰: | -- |
| 载体类型: | 细菌表达 |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | -- |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Cas9-Cys |
| 物种来源: | S. pyogenes |
| 插入大小: | 4173 bp |
| 基因突变: | 额外的半胱氨酸被添加到Cas9的C-末端区域 |
| GenBank编号 | -- |
| 启动子 | T7 Promoter |
| 标签/融合蛋白: |
6X His tag（主干上的N终端）
HA epitope（插入物上的C末端）
NLS（插接件上的C端子）
|
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | Not I(未破坏) |
| 3' 克隆位点: | Not I(未破坏) |
| 5' 测序引物 | TAATACGACTCACTATAGGG |
| 3' 测序引物 | GCTAGTTATTGCTCAGCG |
|
文献引用方法 |
|
| 材料和方法部分: | pET28a/Cas9-Cys was a gift from Hyongbum Kim (Addgene plasmid # 53261 ; http://n2t.net/addgene:53261 ; RRID:Addgene_53261) |
| 参考文献部分: | Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H. Genome Res. 2014 Apr 2. 10.1101/gr.171264.113 PubMed 24696462 |
买家导航
pET28a/Cas9-Cys载体质粒基本信息
pET28a/Cas9-Cys质粒图谱载体图谱和pET28a/Cas9-Cys载体序列质粒序列多克隆位点信息
pET28a/Cas9-Cys质粒载体简介
pET28a/Cas9-Cys质粒序列载体序列
LOCUS pET28a/Cas9-Cys 9546 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses N-terminal His tag fused human codon-optimized Cas9
nuclease having C-terminal Cysteine .
ACCESSION .
VERSION .
KEYWORDS pET28a/Cas9-Cys
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9546)
AUTHORS Ramakrishna S, Kwaku Dad AB, Beloor J, Gopalappa R, Lee SK, Kim H
TITLE Gene disruption by cell-penetrating peptide-mediated delivery of
Cas9 protein and guide RNA.
JOURNAL Genome Res. 2014 Apr 2.
PUBMED 24696462
REFERENCE 2 (bases 1 to 9546)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..9546
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 34..53
/label=T7
/note="T7 promoter, forward primer"
promoter 34..52
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
protein_bind 53..77
/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)."
RBS 92..114
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 133..150
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 160..177
/codon_start=1
/product="thrombin recognition and cleavage site"
/label=thrombin site
/translation="LVPRGS"
CDS 181..213
/codon_start=1
/product="leader peptide from bacteriophage T7 gene 10"
/label=T7 tag (gene 10 leader)
/note="promotes efficient translation in E. coli"
/translation="MASMTGGQQMG"
CDS 256..4359
/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 4375..4395
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 4396..4422
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 4444..4461
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(4514..4532)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 4528..4575
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 4612..5067
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4699..4718)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4909..4930
/label=F1ori-F
/note="F1 origin, forward primer"
CDS complement(5159..5974)
/codon_start=1
/gene="aph(3')-Ia"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin in bacteria or G418
(Geneticin(R)) in eukaryotes"
/translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
primer_bind 5882..5901
/label=Kan-R
/note="Kanamycin resistance gene, reverse primer"
primer_bind 5966..5985
/label=pENTR-R
/note="pENTR vectors, reverse primer"
rep_origin 6096..6684
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6585..6604
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 6838..6855
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 6870..7009
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(6935..6954)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 7095..7117
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(7111..7302)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
CDS complement(8111..9193)
/codon_start=1
/gene="lacI"
/product="lac repressor"
/label=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)."
/translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
ALADSLMQLARQVSRLESGQ"
primer_bind 9155..9174
/label=LacI-R
/note="LacI, reverse primer"
promoter complement(9194..9271)
/gene="lacI"
/label=lacI promoter
primer_bind 9477..9496
/label=pBRrevBam
/note="pBR322 vectors, tet region, downstream of BamHI,
reverse primer"
ORIGIN
1 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt
61 gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc
121 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat
181 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcga gctccgtcga
241 caagcttgcg gccgcatgga caagaagtac agcatcggcc tggacatcgg taccaacagc
301 gtgggctggg ccgtgatcac cgacgagtac aaggtgccca gcaagaagtt caaggtgctg
361 ggcaacaccg accgccacag catcaagaag aacctgatcg gcgccctgct gttcgacagc
421 ggcgagaccg ccgaggccac ccgcctgaag cgcaccgccc gccgccgcta cacccgccgc
481 aagaaccgca tctgctacct gcaggagatc ttcagcaacg agatggccaa ggtggacgac
541 agcttcttcc accgcctgga ggagagcttc ctggtggagg aggacaagaa gcacgagcgc
601 caccccatct tcggcaacat cgtggacgag gtggcctacc acgagaagta ccccaccatc
661 taccacctgc gcaagaagct ggtggacagc accgacaagg ccgacctgcg cctgatctac
721 ctggccctgg cccacatgat caagttccgc ggccacttcc tgatcgaggg cgacctgaac
781 cccgacaaca gcgacgtgga caagctgttc atccagctgg tgcagaccta caaccagctg
841 ttcgaggaga accccatcaa cgccagcggc gtggacgcca aggccatcct gagcgcccgc
901 ctgagcaaga gccgccgcct ggagaacctg atcgcccagc tgcccggcga gaagaagaac
961 ggcctgttcg gcaacctgat cgccctgagc ctgggcctga cccccaactt caagagcaac
1021 ttcgacctgg ccgaggacgc caagctgcag ctgagcaagg acacctacga cgacgacctg
1081 gacaacctgc tggcccagat cggcgaccag tacgccgacc tgttcctggc cgccaagaac
1141 ctgagcgacg ccatcctgct gagcgacatc ctgcgcgtga acaccgagat caccaaggcc
1201 cccctgagcg ccagcatgat caagcgctac gacgagcacc accaggacct gaccctgctg
1261 aaggccctgg tgcgccagca gctgcccgag aagtacaagg agatcttctt cgaccagagc
1321 aagaacggct acgccggcta catcgacggc ggcgccagcc aggaggagtt ctacaagttc
1381 atcaagccca tcctggagaa gatggacggc accgaggagc tgctggtgaa gctgaaccgc
1441 gaggacctgc tgcgcaagca gcgcaccttc gacaacggca gcatccccca ccagatccac
1501 ctgggcgagc tgcacgccat cctgcgccgc caggaggact tctacccctt cctgaaggac
1561 aaccgcgaga agatcgagaa gatcctgacc ttccgcatcc cctactacgt gggccccctg
1621 gcccgcggca acagccgctt cgcctggatg acccgcaaga gcgaggagac catcaccccc
1681 tggaacttcg aggaggtggt ggacaagggc gccagcgccc agagcttcat cgagcgcatg
1741 accaacttcg acaagaacct gcccaacgag aaggtgctgc ccaagcacag cctgctgtac
1801 gagtacttca ccgtgtacaa cgagctgacc aaggtgaagt acgtgaccga gggcatgcgc
1861 aagcccgcct tcctgagcgg cgagcagaag aaggccatcg tggacctgct gttcaagacc
1921 aaccgcaagg tgaccgtgaa gcagctgaag gaggactact tcaagaagat cgagtgcttc
1981 gacagcgtgg agatcagcgg cgtggaggac cgcttcaacg ccagcctggg cacctaccac
2041 gacctgctga agatcatcaa ggacaaggac ttcctggaca acgaggagaa cgaggacatc
2101 ctggaggaca tcgtgctgac cctgaccctg ttcgaggacc gcgagatgat cgaggagcgc
2161 ctgaagacct acgcccacct gttcgacgac aaggtgatga agcagctgaa gcgccgccgc
2221 tacaccggct ggggccgcct gagccgcaag cttatcaacg gcatccgcga caagcagagc
2281 ggcaagacca tcctggactt cctgaagagc gacggcttcg ccaaccgcaa cttcatgcag
2341 ctgatccacg acgacagcct gaccttcaag gaggacatcc agaaggccca ggtgagcggc
2401 cagggcgaca gcctgcacga gcacatcgcc aacctggccg gcagccccgc catcaagaag
2461 ggcatcctgc agaccgtgaa ggtggtggac gagctggtga aggtgatggg ccgccacaag
2521 cccgagaaca tcgtgatcga gatggcccgc gagaaccaga ccacccagaa gggccagaag
2581 aacagccgcg agcgcatgaa gcgcatcgag gagggcatca aggagctggg cagccagatc
2641 ctgaaggagc accccgtgga gaacacccag ctgcagaacg agaagctgta cctgtactac
2701 ctgcagaacg gccgcgacat gtacgtggac caggagctgg acatcaaccg cctgagcgac
2761 tacgacgtgg accacatcgt gccccagagc ttcctgaagg acgacagcat cgacaacaag
2821 gtgctgaccc gcagcgacaa gaaccgcggc aagagcgaca acgtgcccag cgaggaggtg
2881 gtgaagaaga tgaagaacta ctggcgccag ctgctgaacg ccaagctgat cacccagcgc
2941 aagttcgaca acctgaccaa ggccgagcgc ggcggcctga gcgagctgga caaggccggc
3001 ttcatcaagc gccagctggt ggagacccgc cagatcacca agcacgtggc ccagatcctg
3061 gacagccgca tgaacaccaa gtacgacgag aacgacaagc tgatccgcga ggtgaaggtg
3121 atcaccctga agagcaagct ggtgagcgac ttccgcaagg acttccagtt ctacaaggtg
3181 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt ggtgggcacc
3241 gccctgatca agaagtaccc caagctggag agcgagttcg tgtacggcga ctacaaggtg
3301 tacgacgtgc gcaagatgat cgccaagagc gagcaggaga tcggcaaggc caccgccaag
3361 tacttcttct acagcaacat catgaacttc ttcaagaccg agatcaccct ggccaacggc
3421 gagatccgca agcgccccct gatcgagacc aacggcgaga ccggcgagat cgtgtgggac
3481 aagggccgcg acttcgccac cgtgcgcaag gtgctgagca tgccccaggt gaacatcgtg
3541 aagaagaccg aggtgcagac cggcggcttc agcaaggaga gcatcctgcc caagcgcaac
3601 agcgacaagc tgatcgcccg caagaaggac tgggacccca agaagtacgg cggcttcgac
3661 agccccaccg tggcctacag cgtgctggtg gtggccaagg tggagaaggg caagagcaag
3721 aagctgaaga gcgtgaagga gctgctgggc atcaccatca tggagcgcag cagcttcgag
3781 aagaacccca tcgacttcct ggaggccaag ggctacaagg aggtgaagaa ggacctgatc
3841 atcaagctgc ccaagtacag cctgttcgag ctggagaacg gccgcaagcg catgctggcc
3901 agcgccggcg agctgcagaa gggcaacgag ctggccctgc ccagcaagta cgtgaacttc
3961 ctgtacctgg ccagccacta cgagaagctg aagggcagcc ccgaggacaa cgagcagaag
4021 cagctgttcg tggagcagca caagcactac ctggacgaga tcatcgagca gatcagcgag
4081 ttcagcaagc gcgtgatcct ggccgacgcc aacctggaca aggtgctgag cgcctacaac
4141 aagcaccgcg acaagcccat ccgcgagcag gccgagaaca tcatccacct gttcaccctg
4201 accaacctgg gcgcccccgc cgccttcaag tacttcgaca ccaccatcga ccgcaagcgc
4261 tacaccagca ccaaggaggt gctggacgcc accctgatcc accagagcat caccggtctg
4321 tacgagaccc gcatcgacct gagccagctg ggcggcgacg gcggctccgg acctccaaag
4381 aaaaagagaa aagtataccc ctacgacgtg cccgactacg cctgttaagc ggccgcactc
4441 gagcaccacc accaccacca ctgagatccg gctgctaaca aagcccgaaa ggaagctgag
4501 ttggctgctg ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc
4561 ttgaggggtt ttttgctgaa aggaggaact atatccggat tggcgaatgg gacgcgccct
4621 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
4681 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
4741 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
4801 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
4861 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
4921 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
4981 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
5041 ttaacaaaat attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac
5101 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga attaattctt
5161 agaaaaactc atcgagcatc aaatgaaact gcaatttatt catatcagga ttatcaatac
5221 catatttttg aaaaagccgt ttctgtaatg aaggagaaaa ctcaccgagg cagttccata
5281 ggatggcaag atcctggtat cggtctgcga ttccgactcg tccaacatca atacaaccta
5341 ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa atcaccatga gtgacgactg
5401 aatccggtga gaatggcaaa agtttatgca tttctttcca gacttgttca acaggccagc
5461 cattacgctc gtcatcaaaa tcactcgcat caaccaaacc gttattcatt cgtgattgcg
5521 cctgagcgag acgaaatacg cgatcgctgt taaaaggaca attacaaaca ggaatcgaat
5581 gcaaccggcg caggaacact gccagcgcat caacaatatt ttcacctgaa tcaggatatt
5641 cttctaatac ctggaatgct gttttcccgg ggatcgcagt ggtgagtaac catgcatcat
5701 caggagtacg gataaaatgc ttgatggtcg gaagaggcat aaattccgtc agccagttta
5761 gtctgaccat ctcatctgta acatcattgg caacgctacc tttgccatgt ttcagaaaca
5821 actctggcgc atcgggcttc ccatacaatc gatagattgt cgcacctgat tgcccgacat
5881 tatcgcgagc ccatttatac ccatataaat cagcatccat gttggaattt aatcgcggcc
5941 tagagcaaga cgtttcccgt tgaatatggc tcataacacc ccttgtatta ctgtttatgt
6001 aagcagacag ttttattgtt catgaccaaa atcccttaac gtgagttttc gttccactga
6061 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
6121 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
6181 gagccaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
6241 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
6301 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
6361 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
6421 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
6481 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
6541 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
6601 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
6661 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
6721 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
6781 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
6841 gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg
6901 tgcggtattt cacaccgcaa tggtgcactc tcagtacaat ctgctctgat gccgcatagt
6961 taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc cccgacaccc
7021 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca
7081 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg
7141 cgcgaggcag ctgcggtaaa gctcatcagc gtggtcgtga agcgattcac agatgtctgc
7201 ctgttcatcc gcgtccagct cgttgagttt ctccagaagc gttaatgtct ggcttctgat
7261 aaagcgggcc atgttaaggg cggttttttc ctgtttggtc actgatgcct ccgtgtaagg
7321 gggatttctg ttcatggggg taatgatacc gatgaaacga gagaggatgc tcacgatacg
7381 ggttactgat gatgaacatg cccggttact ggaacgttgt gagggtaaac aactggcggt
7441 atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa tgccagcgct tcgttaatac
7501 agatgtaggt gttccacagg gtagccagca gcatcctgcg atgcagatcc ggaacataat
7561 ggtgcagggc gctgacttcc gcgtttccag actttacgaa acacggaaac cgaagaccat
7621 tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag tcgcttcacg ttcgctcgcg
7681 tatcggtgat tcattctgct aaccagtaag gcaaccccgc cagcctagcc gggtcctcaa
7741 cgacaggagc acgatcatgc gcacccgtgg ggccgccatg ccggcgataa tggcctgctt
7801 ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat
7861 tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc
7921 gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt
7981 cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa
8041 ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat
8101 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
8161 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt
8221 tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca
8281 gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg
8341 gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac
8401 caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg
8461 caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac
8521 cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga
8581 gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta
8641 acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt
8701 cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg
8761 ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt
8821 taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt
8881 cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag
8941 atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc
9001 caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca
9061 gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt
9121 tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg
9181 ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac
9241 cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac
9301 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
9361 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
9421 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
9481 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
9541 cgtccg
//
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pET28a/Cas9-Cys质粒载体应用举例
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