| 质粒名称: | pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2A-Puro-WPRE |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 139093 |
| 存储实验室: | Neville Sanjana |
|
载体骨架基本信息 |
|
| 载体名称: | lentiCRISPRv2 |
| 载体出品公司: | Clontech |
| 完整质粒大小: | 14651 bp |
| 载体类型: | 哺乳细胞表达, Lentiviral, CRISPR |
| 筛选标记: | Puromycin |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | dxCas9-NG-VPR |
| 物种来源: | Staphylococcus pyogenes |
| 基因突变: | D10A, H840A, A262T,R324L, S409I, E480K, E543D, M694I, L1111R, D1135V, G1218R, E1219F, A1322R, R1335V,T1337R |
| 克隆方法: | Gibson Cloning |
|
文献引用方法 |
|
| 材料和方法部分: | pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2A-Puro-WPRE was a gift from Neville Sanjana (Addgene plasmid # 139093 ; http://n2t.net/addgene:139093 ; RRID:Addgene_139093) |
| 参考文献部分: | High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation. Legut M, Daniloski Z, Xue X, McKenzie D, Guo X, Wessels HH, Sanjana NE. Cell Rep. 2020 Mar 3;30(9):2859-2868.e5. doi: 10.1016/j.celrep.2020.02.010. 10.1016/j.celrep.2020.02.010 PubMed 32130891 |
pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2
买家导航
pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2载体质粒基本信息
pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2质粒图谱载体图谱和pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2载体序列质粒序列多克隆位点信息
pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2质粒载体简介
pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2质粒序列载体序列
LOCUS pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2A-Puro-WPRE 14651 bp DNA circular SYN 15-DEC-2025
DEFINITION Expresses human codon-optimized inactive xCas9-NG fused to a
transcriptional activator VPR in mammalian cells. For cloning of
sgRNAs using BsmBI. Contains a barcode downstream of sgRNA
cassette..
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 14651)
AUTHORS Legut M, Daniloski Z, Xue X, McKenzie D, Guo X, Wessels HH, Sanjana
NE
TITLE High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene
Knockout and Transcriptional Modulation.
JOURNAL Cell Rep. 2020 Mar 3;30(9):2859-2868.e5. doi:
10.1016/j.celrep.2020.02.010.
PUBMED 32130891
REFERENCE 2 (bases 1 to 14651)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Dec 15, 2025 from SnapGene Server 8.0.1
https://www.biofeng.com
COMMENT SGRef: number: 1; type: "Journal Article"; doi:
"10.1016/j.celrep.2020.02"; journalName: "Cell Rep"; date:
"2020-03-3- 3"; volume: "30"; issue: "9"; pages: "2859-2868.e5"
COMMENT Sequence Label: pCC_08 -
hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCa...
FEATURES Location/Qualifiers
source 1..14651
/mol_type="other DNA"
/organism="synthetic DNA construct"
primer_bind complement(47..66)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
enhancer 238..617
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 619..817
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 772..792
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
LTR 835..1015
/label=5' LTR (truncated)
/note="truncated 5' long terminal repeat (LTR) from HIV-1"
misc_feature 1062..1187
/label=HIV-1 Psi
/note="packaging signal of human immunodeficiency virus
type 1"
misc_feature 1680..1913
/label=RRE
/note="The Rev response element (RRE) of HIV-1 allows for
Rev-dependent mRNA export from the nucleus to the
cytoplasm."
CDS 2098..2142
/codon_start=1
/product="antigenic peptide corresponding to amino acids
655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
et al., 2013)"
/label=gp41 peptide
/note="recognized by the 2H10 single-chain llama nanobody"
/translation="KNEQELLELDKWASL"
misc_feature 2440..2557
/label=cPPT/CTS
/note="central polypurine tract and central termination
sequence of HIV-1"
promoter 2608..2856
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA
(Domitrovich & Kunkel, 2003)"
primer_bind 2608..2628
/label=hU6-F
/note="Human U6 promoter, forward primer"
primer_bind 2779..2798
/label=LKO.1 5'
/note="Human U6 promoter, forward primer"
promoter 3027..3238
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 3257..3266
/label=Kozak sequence
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
/regulatory_class="other"
CDS 3263..7366
/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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFI
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 7379..7399
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
protein_bind 7436..7460
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 7490..7639
/codon_start=1
/product="tetrameric repeat of the minimal activation
domain of herpes simplex virus VP16 (Beerli et al., 1998)"
/label=VP64
/translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
DLDML"
CDS 7664..7684
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 8114..8470
/codon_start=1
/product="transcriptional activation domain of human RelA,
also known as p65 (O'Shea and Perkins, 2008)"
/label=RelA (p65) AD
/translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
EDFSSIADMDFSALL"
CDS 8489..9058
/codon_start=1
/product="transcriptional activation domain from the human
herpesvirus 4 (Epstein-Barr virus) replication and
transcription activator Rta/BRLF1 (Hardwick et al., 1992;
Chavez et al., 2015)"
/label=Rta AD
/translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
DTFLNDECLLHAMHISTGLSIFDTSLF"
CDS 9074..9130
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 9131..9727
/codon_start=1
/gene="pac from Streptomyces alboniger"
/product="puromycin N-acetyltransferase"
/label=PuroR
/note="confers resistance to puromycin"
/translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
primer_bind 9624..9644
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 9743..10331
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(9796..9816)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(10214..10225)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 10403..10636
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(10662..10679)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 10668..10892
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 10938..11366
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(11025..11044)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 11235..11256
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(11375..11395)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 11380..11709
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
rep_origin 11560..11695
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 11622..11641
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 11757..11804
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 11823..12197
/codon_start=1
/gene="Sh ble from Streptoalloteichus hindustanus"
/product="antibiotic-binding protein"
/label=BleoR
/note="confers resistance to bleomycin, phleomycin, and
Zeocin(TM)"
/translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
EFALRDPAGNCVHFVAEEQD"
polyA_signal 12327..12448
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(12364..12383)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 12418..12437
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(12497..12513)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(12497..12513)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(12510..12532)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 12521..12537
/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(join(12545..12551,12552..12569,12570..12575))
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 12590..12611
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(12728..12745)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(12899..13487)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(12979..12998)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(join(13658..14449,14450..14518))
/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 14281..14300
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(14519..14623)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
1501 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
1561 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
1621 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
1681 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
1741 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
1801 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
1861 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
1921 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
1981 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
2041 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
2101 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
2161 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
2221 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
2281 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
2341 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc ggcactgcgt
2401 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
2461 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
2521 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
2581 agatccagtt tggttaatta aggtaccgag ggcctatttc ccatgattcc ttcatatttg
2641 catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag
2701 atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta
2761 aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc
2821 ttggctttat atatcttgtg gaaaggacga aacaccggag acggacgtct ctgtttaaga
2881 gctatgctgg aaacagcata gcaagtttaa ataaggctag tccgttatca acttgaaaaa
2941 gtggcaccga gtcggtgctt tttttgaatt ctttatggct agctaggtct tgaaaggagt
3001 gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
3061 gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg
3121 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
3181 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacagga
3241 ccggttctag agcgctgcca ccatggacaa gaagtacagc atcggcctgg ccatcggcac
3301 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa
3361 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt
3421 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac
3481 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt
3541 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca
3601 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc
3661 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct
3721 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga
3781 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa
3841 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc
3901 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa
3961 gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa
4021 gagcaacttc gacctggccg aggataccaa actgcagctg agcaaggaca cctacgacga
4081 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc
4141 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac
4201 caaggccccc ctgagcgcct ctatgatcaa gctgtacgac gagcaccacc aggacctgac
4261 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga
4321 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta
4381 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct
4441 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcatca tcccccacca
4501 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct
4561 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg
4621 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat
4681 caccccctgg aacttcgaga aggtggtgga caagggcgct tccgcccaga gcttcatcga
4741 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct
4801 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg
4861 aatgagaaag cccgccttcc tgagcggcga ccagaaaaag gccatcgtgg acctgctgtt
4921 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga
4981 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac
5041 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga
5101 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga
5161 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg
5221 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa
5281 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt
5341 catccagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt
5401 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat
5461 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg
5521 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg
5581 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag
5641 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct
5701 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct
5761 gtccgactac gatgtggacg ctatcgtgcc tcagagcttt cttaaggacg actccatcga
5821 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga
5881 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac
5941 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa
6001 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca
6061 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt
6121 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta
6181 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt
6241 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta
6301 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac
6361 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc
6421 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt
6481 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa
6541 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tccggcccaa
6601 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg
6661 cttcgttagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa
6721 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag
6781 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga
6841 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat
6901 gctggcctct gcccggttcc tgcagaaggg aaacgaactg gccctgccct ccaaatatgt
6961 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga
7021 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat
7081 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc
7141 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt
7201 taccctgacc aatctgggag cccctcgggc cttcaagtac tttgacacca ccatcgaccg
7261 gaaggtgtac cggagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac
7321 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacagca gggctgaccc
7381 caagaagaag aggaaggtgt cgccagggat ccgtcgactt gacgcgttga tatcaacaag
7441 tttgtacaaa aaagcaggct acaaagaggc cagcggttcc ggacgggctg acgcattgga
7501 cgattttgat ctggatatgc tgggaagtga cgccctcgat gattttgacc ttgacatgct
7561 tggttcggat gcccttgatg actttgacct cgacatgctc ggcagtgacg cccttgatga
7621 tttcgacctg gacatgctga ttaactctag aagttccgga tctccgaaaa agaaacgcaa
7681 agttggtagc cagtacctgc ccgacaccga cgaccggcac cggatcgagg aaaagcggaa
7741 gcggacctac gagacattca agagcatcat gaagaagtcc cccttcagcg gccccaccga
7801 ccctagacct ccacctagaa gaatcgccgt gcccagcaga tccagcgcca gcgtgccaaa
7861 acctgccccc cagccttacc ccttcaccag cagcctgagc accatcaact acgacgagtt
7921 ccctaccatg gtgttcccca gcggccagat ctctcaggcc tctgctctgg ctccagcccc
7981 tcctcaggtg ctgcctcagg ctcctgctcc tgcaccagct ccagccatgg tgtctgcact
8041 ggctcaggca ccagcacccg tgcctgtgct ggctcctgga cctccacagg ctgtggctcc
8101 accagcccct aaacctacac aggccggcga gggcacactg tctgaagctc tgctgcagct
8161 gcagttcgac gacgaggatc tgggagccct gctgggaaac agcaccgatc ctgccgtgtt
8221 caccgacctg gccagcgtgg acaacagcga gttccagcag ctgctgaacc agggcatccc
8281 tgtggcccct cacaccaccg agcccatgct gatggaatac cccgaggcca tcacccggct
8341 cgtgacaggc gctcagaggc ctcctgatcc agctcctgcc cctctgggag caccaggcct
8401 gcctaatgga ctgctgtctg gcgacgagga cttcagctct atcgccgata tggatttctc
8461 agccttgctg ggctctggca gcggcagccg ggattccagg gaagggatgt ttttgccgaa
8521 gcctgaggcc ggctccgcta ttagtgacgt gtttgagggc cgcgaggtgt gccagccaaa
8581 acgaatccgg ccatttcatc ctccaggaag tccatgggcc aaccgcccac tccccgccag
8641 cctcgcacca acaccaaccg gtccagtaca tgagccagtc gggtcactga ccccggcacc
8701 agtccctcag ccactggatc cagcgcccgc agtgactccc gaggccagtc acctgttgga
8761 ggatcccgat gaagaaacga gccaggctgt caaagccctt cgggagatgg ccgatactgt
8821 gattccccag aaggaagagg ctgcaatctg tggccaaatg gacctttccc atccgccccc
8881 aaggggccat ctggatgagc tgacaaccac acttgagtcc atgaccgagg atctgaacct
8941 ggactcaccc ctgaccccgg aattgaacga gattctggat accttcctga acgacgagtg
9001 cctcttgcat gccatgcata tcagcacagg actgtccatc ttcgacacat ctctctttga
9061 attcggatcc ggcgcaacaa acttctctct gctgaaacaa gccggagatg tcgaagagaa
9121 tcctggaccg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
9181 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
9241 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
9301 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
9361 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
9421 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
9481 caaggagccc gcgtggttcc tggccaccgt cggagtctcg cccgaccacc agggcaaggg
9541 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
9601 cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
9661 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
9721 tgcctgaacg cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac
9781 tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt
9841 gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt
9901 gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt
9961 gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg
10021 gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg
10081 ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc
10141 atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt
10201 ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc
10261 tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc
10321 cgcctccccg cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac
10381 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg
10441 ctttttgctt gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
10501 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
10561 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
10621 tggaaaatct ctagcagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag
10681 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
10741 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
10801 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
10861 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
10921 ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
10981 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
11041 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
11101 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
11161 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
11221 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
11281 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
11341 gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga
11401 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
11461 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
11521 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
11581 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
11641 gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
11701 ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgttg
11761 acaattaatc atcggcatag tatatcggca tagtataata cgacaaggtg aggaactaaa
11821 ccatggccaa gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg
11881 tcgagttctg gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg
11941 gtgtggtccg ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg
12001 acaacaccct ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg
12061 aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc
12121 agccgtgggg gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg
12181 ccgaggagca ggactgacac gtgctacgag atttcgattc caccgccgcc ttctatgaaa
12241 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
12301 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
12361 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
12421 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
12481 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
12541 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
12601 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
12661 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
12721 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
12781 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
12841 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
12901 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
12961 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
13021 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
13081 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
13141 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
13201 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
13261 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
13321 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
13381 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
13441 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
13501 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
13561 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
13621 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
13681 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
13741 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
13801 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
13861 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
13921 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
13981 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
14041 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
14101 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
14161 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
14221 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
14281 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
14341 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
14401 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
14461 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
14521 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
14581 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
14641 tgccacctga c
//
pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2载体图谱质粒图谱pdf版和pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2载体序列质粒序列等相关资料下载
pCC_08 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dxCas9NG-NLS-VPR-2质粒载体应用举例
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