| 质粒名称: | LCV2_mClover3_AAVS1_sgRNA_2 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 155099 |
| 存储实验室: | Jason Moffat |
|
载体骨架基本信息 |
|
| 载体名称: | LCV2_mClover3 |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 14753 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 哺乳动物表达，慢病毒，CRISPR |
| 筛选标记: | 嘌呤霉素 |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 30 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | AAVS1_sgRNA_2 |
| 物种来源: | H. sapiens (human) |
| 插入大小: | 20 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | U6 promoter |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5' 克隆位点: | BsmBI/Esp3I（在克隆过程中被破坏） |
| 3' 克隆位点: | BsmBI/Esp3I（在克隆过程中被破坏） |
| 5' 测序引物 | ACTATCATATGCTTACCGTAAC |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | LCV2_mClover3_AAVS1_sgRNA_2 was a gift from Jason Moffat (Addgene plasmid # 155099 ; http://n2t.net/addgene:155099 ; RRID:Addgene_155099) |
| 参考文献部分: | Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism. Aregger M, Lawson KA, Billmann M, Costanzo M, Tong AHY, Chan KA, Rahman M, Brown KR, Ross C, Usaj M, Nedyalkova L, Sizova O, Habsid A, Pawling J, Lin ZY, Abdouni H, Wong C, Weiss A, Mero P, Dennis JW, Gingras A, Myers CL, Andrews BJ, Boone C, Moffat J. Nat Metab (2020) 10.1038/s42255-020-0211-z |
买家导航
LCV2_mClover3_AAVS1_sgRNA_2载体质粒基本信息
LCV2_mClover3_AAVS1_sgRNA_2质粒图谱载体图谱和LCV2_mClover3_AAVS1_sgRNA_2载体序列质粒序列多克隆位点信息
LCV2_mClover3_AAVS1_sgRNA_2质粒载体简介
LCV2_mClover3_AAVS1_sgRNA_2质粒序列载体序列
LOCUS LCV2_mClover3_AAVS1_sgRNA_2 13724 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION lentiviral plasmid expressing mClover3, Cas9 and a gRNA targeting
the AAVS1 safe harbor locus.
ACCESSION .
VERSION .
KEYWORDS LCV2_mClover3_AAVS1_sgRNA_2
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 13724)
AUTHORS Aregger M, Lawson KA, Billmann M, Costanzo M, Tong AHY, Chan KA,
Rahman M, Brown KR, Ross C, Usaj M, Nedyalkova L, Sizova O, Habsid
A, Pawling J, Lin ZY, Abdouni H, Wong C, Weiss A, Mero P, Dennis JW,
Gingras A, Myers CL, Andrews BJ, Boone C, Moffat J
TITLE Systematic mapping of genetic interactions for de novo fatty acid
synthesis identifies C12orf49 as a regulator of lipid metabolism
JOURNAL Nat Metab (2020)
REFERENCE 2 (bases 1 to 13724)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..13724
/organism="synthetic DNA construct"
/mol_type="other DNA"
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..2848
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
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"
misc_RNA 2878..2953
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
promoter 3015..3226
/label=EF-1-alpha core promoter
/note="core promoter for human elongation factor
EF-1-alpha"
regulatory 3245..3254
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 3251..7354
/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 7355..7402
/codon_start=1
/product="bipartite nuclear localization signal from
nucleoplasmin"
/label=nucleoplasmin NLS
/translation="KRPAATKKAGQAKKKK"
CDS 7403..7426
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 7436..7489
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
CDS 7496..8209
/codon_start=1
/product="bright green-yellow fluorescent protein derived
from mClover2, with improved photostability (Bajar et al.,
2003)"
/label=mClover3
/note="mammalian codon-optimized"
/translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIKA
NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLLE
FVTAAGITHGMDELYK"
primer_bind complement(7538..7559)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind 8146..8167
/label=EGFP-C
/note="EGFP, forward primer"
CDS 8219..8275
/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 8276..8872
/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 8769..8789
/label=Puro-F
/note="Puromycin resistance gene, forward primer"
misc_feature 8888..9476
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(8941..8961)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(9359..9370)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
LTR 9548..9781
/label=3' LTR (Delta-U3)
/note="self-inactivating 3' long terminal repeat (LTR) from
HIV-1"
primer_bind complement(9807..9824)
/label=BGH-rev
/note="Bovine growth hormone terminator, reverse primer.
Also called BGH reverse"
polyA_signal 9813..10037
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
rep_origin 10083..10511
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(10170..10189)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 10380..10401
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(10520..10540)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
promoter 10564..10774
/label=SV40 promoter
/note="SV40 early promoter"
promoter 10586..10782
/label=SV40 promoter
/note="SV40 early promoter"
rep_origin 10633..10768
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 10695..10714
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
promoter 10830..10877
/label=EM7 promoter
/note="synthetic bacterial promoter "
CDS 10896..11270
/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 11400..11521
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(11437..11456)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 11491..11510
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind complement(11570..11586)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(11570..11586)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(11583..11605)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 11594..11610
/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(11618..11648)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 11663..11684
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(11801..11818)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(11972..12560)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(12052..12071)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(12731..13591)
/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 13354..13373
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(13592..13696)
/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 aacaccggtc accaatcctg tccctaggtt
2881 ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc
2941 accgagtcgg tgcttttttg aattcgctag ctaggtcttg aaaggagtgg gaattggctc
3001 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
3061 ggtcggcaat tgatccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
3121 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
3181 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc ggttctagag
3241 cgctgccacc atggacaaga agtacagcat cggcctggac atcggcacca actctgtggg
3301 ctgggccgtg atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa
3361 caccgaccgg cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga
3421 aacagccgag gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa
3481 ccggatctgc tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt
3541 cttccacaga ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc
3601 catcttcggc aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca
3661 cctgagaaag aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc
3721 cctggcccac atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga
3781 caacagcgac gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga
3841 ggaaaacccc atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag
3901 caagagcaga cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct
3961 gttcggaaac ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga
4021 cctggccgag gatgccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa
4081 cctgctggcc cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc
4141 cgacgccatc ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct
4201 gagcgcctct atgatcaaga gatacgacga gcaccaccag gacctgaccc tgctgaaagc
4261 tctcgtgcgg cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa
4321 cggctacgcc ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa
4381 gcccatcctg gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga
4441 cctgctgcgg aagcagcgga ccttcgacaa cggcagcatc ccccaccaga tccacctggg
4501 agagctgcac gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg
4561 ggaaaagatc gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag
4621 gggaaacagc agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa
4681 cttcgaggaa gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa
4741 cttcgataag aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta
4801 cttcaccgtg tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc
4861 cgccttcctg agcggcgagc agaaaaaggc catcgtggac ctgctgttca agaccaaccg
4921 gaaagtgacc gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc
4981 cgtggaaatc tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct
5041 gctgaaaatt atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga
5101 agatatcgtg ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa
5161 aacctatgcc cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac
5221 cggctggggc aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa
5281 gacaatcctg gatttcctga agtccgacgg cttcgccaac agaaacttca tgcagctgat
5341 ccacgacgac agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg
5401 cgatagcctg cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat
5461 cctgcagaca gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga
5521 gaacatcgtg atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag
5581 ccgcgagaga atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa
5641 agaacacccc gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca
5701 gaatgggcgg gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga
5761 tgtggaccat atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct
5821 gaccagaagc gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa
5881 gaagatgaag aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt
5941 cgacaatctg accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat
6001 caagagacag ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc
6061 ccggatgaac actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac
6121 cctgaagtcc aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga
6181 gatcaacaac taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct
6241 gatcaaaaag taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga
6301 cgtgcggaag atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt
6361 cttctacagc aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat
6421 ccggaagcgg cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg
6481 ccgggatttt gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa
6541 gaccgaggtg cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga
6601 taagctgatc gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc
6661 caccgtggcc tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact
6721 gaagagtgtg aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa
6781 tcccatcgac tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa
6841 gctgcctaag tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc
6901 cggcgaactg cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta
6961 cctggccagc cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct
7021 gtttgtggaa cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc
7081 caagagagtg atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca
7141 ccgggataag cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa
7201 tctgggagcc cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac
7261 cagcaccaaa gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga
7321 gacacggatc gacctgtctc agctgggagg cgacaagcga cctgccgcca caaagaaggc
7381 tggacaggct aagaagaaga aagattacaa agacgatgac gataagggca gtggagaggg
7441 cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccac tcgaggtgag
7501 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
7561 aaacggccac aagttcagcg tccgcggcga gggcgagggc gatgccacca acggcaagct
7621 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
7681 caccttcggc tacggcgtgg cctgcttcag ccgctacccc gaccacatga agcagcacga
7741 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct ctttcaagga
7801 cgacggtacc tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
7861 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
7921 gtacaacttc aacagccact acgtctatat cacggccgac aagcagaaga actgcatcaa
7981 ggctaacttc aagatccgcc acaacgttga ggacggcagc gtgcagctcg ccgaccacta
8041 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
8101 ccatcagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
8161 gttcgtgacc gccgccggga ttacacatgg catggacgag ctgtacaagg gatccggcgc
8221 aacaaacttc tctctgctga aacaagccgg agatgtcgaa gagaatcctg gaccgaccga
8281 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
8341 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
8401 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
8461 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
8521 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
8581 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
8641 gttcctggcc accgtcggag tctcgcccga ccaccagggc aagggtctgg gcagcgccgt
8701 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
8761 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
8821 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta
8881 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
8941 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
9001 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
9061 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
9121 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
9181 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
9241 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
9301 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
9361 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
9421 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
9481 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
9541 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
9601 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
9661 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
9721 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
9781 agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
9841 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
9901 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
9961 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
10021 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
10081 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
10141 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
10201 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
10261 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
10321 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
10381 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
10441 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
10501 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
10561 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc
10621 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
10681 tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag
10741 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt
10801 atatccattt tcggatctga tcagcacgtg ttgacaatta atcatcggca tagtatatcg
10861 gcatagtata atacgacaag gtgaggaact aaaccatggc caagttgacc agtgccgttc
10921 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt
10981 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt
11041 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc
11101 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg
11161 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc
11221 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga cacgtgctac
11281 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
11341 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
11401 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
11461 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
11521 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
11581 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
11641 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
11701 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
11761 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
11821 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
11881 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
11941 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
12001 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
12061 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
12121 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
12181 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
12241 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
12301 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
12361 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
12421 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
12481 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
12541 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
12601 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
12661 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
12721 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
12781 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
12841 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
12901 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
12961 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
13021 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
13081 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
13141 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
13201 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
13261 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
13321 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
13381 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
13441 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
13501 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
13561 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
13621 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
13681 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgac
//
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LCV2_mClover3_AAVS1_sgRNA_2质粒载体应用举例
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