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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
GeneArt CRISPR Nuclease Vector-OFP-gRNA
- 库存：
100
- 供应商：
上海烜雅生物科技有限公司
- 规格：
干粉/液体
基本信息
名称：GeneArt CRISPR Nuclease Vector-OFP-gRNA人源基因引导质粒
别称: GeneArt CRISPR Nuclease Vector-OFP-gRNA
原核抗性: Amp
克隆菌株: DH5a
培养条件: 37度
质粒属性
载体宿主：哺乳细胞
载体用途：基因敲除
基因种属：人
基因类型：CRISPR
原核抗性：Amp
真核抗性：
荧光蛋白：
质粒简介
质粒由上海烜雅生物科技有限公司提供
质粒图谱
质粒序列
LOCUS Exported 9227 bp ds-DNA circular SYN 29-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9227)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..9227
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 50..429
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 430..633
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
regulatory 728..737
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 737..778
/codon_start=1
/product="epitope tag from simian virus 5"
/label=V5 tag
/translation="GKPIPNPLLGLDST"
CDS 779..826
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="APKKKRKVGIHGVPAA"
CDS 827..4927
/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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLS-DAILLS-DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLS-DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
RBS 4840..4848
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 4928..4960
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="SRADPKKKRKV"
CDS 4982..5035
/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 5036..5713
/codon_start=1
/label=OFP
/translation="MNLSKNVSVSVYMKGNVNNHEFEYDGEGGGDPYTGKYSMKMTLRG
QNSLPFSYDIITTAFQYGFRVFTKYPEGIVDYFKDSLPDAFQWNRRIVFEDGGVLNMSS
DITYKDNVLHGDVKAEGVNFPPNGPVMKNEIVMEEPTEETFTPKNGVLVGFCPKAYLLK
DGSYYYGNMTTFYRSKKSGQAPPGYHFVKHRLVKTNVGHGFKTVEQTEYATAHVSDLPK
FEA"
polyA_signal 5785..5833
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 6035..6463
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6483..6723
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 6728..6732
/label=overhang
misc_feature 6733..6759
/label=crRNA
misc_RNA 6760..6835
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 6760..6764
/label=overhang
misc_feature 6836..6841
/label=PolIII transcription terminator
rep_origin complement(7287..7875)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8046..8906)
/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"
promoter complement(8907..9011)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
721 agcggccgcc accatgggca agcccatccc taaccccctg ttggggctgg acagcaccgc
781 tcccaaaaag aaaaggaagg tgggcattca cggcgtgcct gcggccgaca aaaagtacag
841 catcggcctt gatatcggca ccaatagcgt gggctgggcc gttatcacag acgaatacaa
901 ggtacccagc aagaagttca aggtgctggg gaatacagac aggcactcta tcaagaaaaa
961 ccttatcggg gctctgctgt ttgactcagg cgagaccgcc gaggccacca ggttgaagag
1021 gaccgcaagg cgaaggtaca cccggaggaa gaacaggatc tgctatctgc aggagatctt
1081 cagcaacgag atggccaagg tggacgacag cttcttccac aggctggagg agagcttcct
1141 tgtcgaggag gataagaagc acgaacgaca ccccatcttc ggcaacatag tcgacgaggt
1201 cgcttatcac gagaagtacc ccaccatcta ccacctgcga aagaaattgg tggatagcac
1261 cgataaagcc gacttgcgac ttatctactt ggctctggcg cacatgatta agttcagggg
1321 ccacttcctg atcgagggcg accttaaccc cgacaacagt gacgtagaca aattgttcat
1381 ccagcttgta cagacctata accagctgtt cgaggaaaac cctattaacg ccagcggggt
1441 ggatgcgaag gccatactta gcgccaggct gagcaaaagc aggcgcttgg agaacctgat
1501 agcccagctg cccggtgaaa agaagaacgg cctcttcggt aatctgattg ccctgagcct
1561 gggcctgacc cccaacttca agagcaactt cgacctggca gaagatgcca agctgcagtt
1621 gagtaaggac acctatgacg acgacttgga caatctgctc gcccaaatcg gcgaccagta
1681 cgctgacctg ttcctcgccg ccaagaacct ttctgacgca atcctgctta gcgatatcct
1741 tagggtgaac acagagatca ccaaggcccc cctgagcgcc agcatgatca agaggtacga
1801 cgagcaccat caggacctga cccttctgaa ggccctggtg aggcagcaac tgcccgagaa
1861 gtacaaggag atctttttcg accagagcaa gaacggctac gccggctaca tcgacggcgg
1921 agccagccaa gaggagttct acaagttcat caagcccatc ctggagaaga tggatggcac
1981 cgaggagctg ctggtgaagc tgaacaggga agatttgctc cggaagcaga ggacctttga
2041 caacggtagc atcccccacc agatccacct gggcgagctg cacgcaatac tgaggcgaca
2101 ggaggatttc taccccttcc tcaaggacaa tagggagaaa atcgaaaaga ttctgacctt
2161 caggatcccc tactacgtgg gccctcttgc caggggcaac agccgattcg cttggatgac
2221 aagaaagagc gaggagacca tcaccccctg gaacttcgag gaagtggtgg acaaaggagc
2281 aagcgcgcag tctttcatcg aacggatgac caatttcgac aaaaacctgc ctaacgagaa
2341 ggtgctgccc aagcacagcc tgctttacga gtacttcacc gtgtacaacg agctcaccaa
2401 ggtgaaatat gtgaccgagg gcatgcgaaa acccgctttc ctgagcggcg agcagaagaa
2461 ggccatcgtg gacctgctgt tcaagaccaa caggaaggtg accgtgaagc agctgaagga
2521 ggactacttc aagaagatcg agtgctttga tagcgtggaa ataagcggcg tggaggacag
2581 gttcaacgcc agcctgggca cctaccacga cttgttgaag ataatcaaag acaaggattt
2641 cctggataat gaggagaacg aggatatact cgaggacatc gtgctgactt tgaccctgtt
2701 tgaggaccga gagatgattg aagaaaggct caaaacctac gcccacctgt tcgacgacaa
2761 agtgatgaaa caactgaaga gacgaagata caccggctgg ggcagactgt ccaggaagct
2821 catcaacggc attagggaca agcagagcgg caagaccatc ctggatttcc tgaagtccga
2881 cggcttcgcc aaccgaaact tcatgcagct gattcacgat gacagcttga ccttcaagga
2941 ggacatccag aaggcccagg ttagcggcca gggcgactcc ctgcacgaac atattgcaaa
3001 cctggcaggc tcccctgcga tcaagaaggg catactgcag accgttaagg ttgtggacga
3061 attggtcaag gtcatgggca ggcacaagcc cgaaaacata gttatagaga tggccagaga
3121 gaaccagacc acccaaaagg gccagaagaa cagccgggag cgcatgaaaa ggatcgagga
3181 gggtatcaag gaactcggaa gccagatcct caaagagcac cccgtggaga atacccagct
3241 ccagaacgag aagctgtacc tgtactacct gcagaacggc agggacatgt acgttgacca
3301 ggagttggac atcaacaggc tttcagacta tgacgtggat cacatagtgc cccagagctt
3361 tcttaaagac gatagcatcg acaacaaggt cctgacccgc tccgacaaaa acaggggcaa
3421 aagcgacaac gtgccaagcg aagaggtggt taaaaagatg aagaactact ggaggcaact
3481 gctcaacgcg aaattgatca cccagagaaa gttcgataac ctgaccaagg ccgagagggg
3541 cggactctcc gaacttgaca aagcgggctt cataaagagg cagctggtcg agacccgaca
3601 gatcacgaag cacgtggccc aaatcctcga cagcagaatg aataccaagt acgatgagaa
3661 tgacaaactc atcagggaag tgaaagtgat taccctgaag agcaagttgg tgtccgactt
3721 tcgcaaagat ttccagttct acaaggtgag ggagatcaac aactaccacc atgcccacga
3781 cgcatacctg aacgccgtgg tcggcaccgc cctgattaag aagtatccaa agctggagtc
3841 cgaatttgtc tacggcgact acaaagttta cgatgtgagg aagatgatcg ctaagagcga
3901 acaggagatc ggcaaggcca ccgctaagta tttcttctac agcaacatca tgaacttttt
3961 caagaccgag atcacacttg ccaacggcga aatcaggaag aggccgctta tcgagaccaa
4021 cggtgagacc ggcgagatcg tgtgggacaa gggcagggac ttcgccaccg tgaggaaagt
4081 cctgagcatg ccccaggtga atattgtgaa aaaaactgag gtgcagacag gcggctttag
4141 caaggaatcc atcctgccca agaggaacag cgacaagctg atcgcccgga agaaggactg
4201 ggaccctaag aagtatggag gcttcgacag ccccaccgta gcctacagcg tgctggtggt
4261 cgcgaaggta gagaagggga agagcaagaa actgaagagc gtgaaggagc tgctcggcat
4321 aaccatcatg gagaggtcca gctttgagaa gaaccccatt gactttttgg aagccaaggg
4381 ctacaaagag gtcaaaaagg acctgatcat caaactcccc aagtactccc tgtttgaatt
4441 ggagaacggc agaaagagga tgctggcgag cgctggggaa ctgcaaaagg gcaacgaact
4501 ggcgctgccc agcaagtacg tgaattttct gtacctggcg tcccactacg aaaagctgaa
4561 aggcagcccc gaggacaacg agcagaagca gctgttcgtg gagcagcaca agcattacct
4621 ggacgagata atcgagcaaa tcagcgagtt cagcaagagg gtgattctgg ccgacgcgaa
4681 cctggataag gtcctcagcg cctacaacaa gcaccgagac aaacccatca gggagcaggc
4741 cgagaatatc atacacctgt tcaccctgac aaatctgggc gcacctgcgg cattcaaata
4801 cttcgatacc accatcgaca ggaaaaggta cactagcact aaggaggtgc tggatgccac
4861 cttgatccac cagtccatta ccggcctgta tgagaccagg atcgacctga gccagcttgg
4921 aggcgactct agggcggacc caaaaaagaa aaggaaggtg gaattctcta gaggcagtgg
4981 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaatgaa
5041 cctgagcaaa aacgtgagcg tgagcgtgta tatgaagggg aacgtcaaca atcatgagtt
5101 tgagtacgac ggggaaggtg gtggtgatcc ttatacaggt aaatattcca tgaagatgac
5161 gctacgtggt caaaattccc tacccttttc ctatgatatc attaccacgg catttcagta
5221 tggtttccgc gtatttacaa aataccctga gggaattgtt gactatttta aggactcgct
5281 tcccgacgca ttccagtgga acagacgaat tgtgtttgaa gatggtggag tactaaacat
5341 gagcagtgat atcacatata aagataatgt tctgcatggt gacgtcaagg ctgagggagt
5401 gaacttcccg ccgaatgggc cagtgatgaa gaatgaaatt gtgatggagg aaccgactga
5461 agaaacattt actccaaaaa acggggttct tgttggcttt tgtcccaaag cgtacttact
5521 taaagacggt tcctattact atggaaatat gacaacattt tacagatcca agaaatctgg
5581 ccaggcacct cctgggtatc actttgttaa gcatcgtctc gtcaagacca atgtgggaca
5641 tggatttaag acggttgagc agactgaata tgccactgct catgtcagtg atcttcccaa
5701 gttcgaagct tgataatgag tttaaacggg ggaggctaac tgaaacacgg aaggagacaa
5761 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacgggtgtt
5821 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
5881 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
5941 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagca
6001 gatctgcgca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
6061 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
6121 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
6181 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
6241 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
6301 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
6361 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
6421 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattaag gtcgggcagg
6481 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
6541 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
6601 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
6661 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
6721 gacgaaacac cgtcaagagg aacaagcttt actcaagagg ttttagagct agaaatagca
6781 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
6841 tctagtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
6901 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
6961 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
7021 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
7081 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
7141 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
7201 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
7261 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
7321 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
7381 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
7441 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
7501 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
7561 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
7621 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
7681 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
7741 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
7801 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
7861 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
7921 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
7981 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
8041 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
8101 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
8161 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
8221 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
8281 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
8341 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
8401 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
8461 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
8521 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
8581 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
8641 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
8701 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
8761 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
8821 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
8881 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
8941 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
9001 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
9061 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
9121 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
9181 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagg
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
名称：GeneArt CRISPR Nuclease Vector-OFP-gRNA人源基因引导质粒
别称: GeneArt CRISPR Nuclease Vector-OFP-gRNA
原核抗性: Amp
克隆菌株: DH5a
培养条件: 37度
质粒属性
载体宿主：哺乳细胞
载体用途：基因敲除
基因种属：人
基因类型：CRISPR
原核抗性：Amp
真核抗性：
荧光蛋白：
质粒简介
质粒由上海烜雅生物科技有限公司提供
质粒图谱
质粒序列
LOCUS Exported 9227 bp ds-DNA circular SYN 29-SEP-2017
DEFINITION synthetic circular DNA
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9227)
AUTHORS .
TITLE Direct Submission
http://www.miaolingbio.com
FEATURES Location/Qualifiers
source 1..9227
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 50..429
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 430..633
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
regulatory 728..737
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 737..778
/codon_start=1
/product="epitope tag from simian virus 5"
/label=V5 tag
/translation="GKPIPNPLLGLDST"
CDS 779..826
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="APKKKRKVGIHGVPAA"
CDS 827..4927
/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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLS-DAILLS-DILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLS-DYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
RBS 4840..4848
/label=Shine-Dalgarno sequence
/note="full consensus sequence for ribosome-binding sites
upstream of start codons in E. coli; complementary to a
region in the 3' end of the 16S rRNA (Chen et al., 1994)"
CDS 4928..4960
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="SRADPKKKRKV"
CDS 4982..5035
/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 5036..5713
/codon_start=1
/label=OFP
/translation="MNLSKNVSVSVYMKGNVNNHEFEYDGEGGGDPYTGKYSMKMTLRG
QNSLPFSYDIITTAFQYGFRVFTKYPEGIVDYFKDSLPDAFQWNRRIVFEDGGVLNMSS
DITYKDNVLHGDVKAEGVNFPPNGPVMKNEIVMEEPTEETFTPKNGVLVGFCPKAYLLK
DGSYYYGNMTTFYRSKKSGQAPPGYHFVKHRLVKTNVGHGFKTVEQTEYATAHVSDLPK
FEA"
polyA_signal 5785..5833
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 6035..6463
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 6483..6723
/label=U6 promoter
/note="RNA polymerase III promoter for human U6 snRNA"
misc_feature 6728..6732
/label=overhang
misc_feature 6733..6759
/label=crRNA
misc_RNA 6760..6835
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
misc_feature 6760..6764
/label=overhang
misc_feature 6836..6841
/label=PolIII transcription terminator
rep_origin complement(7287..7875)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(8046..8906)
/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"
promoter complement(8907..9011)
/gene="bla"
/label=AmpR promoter
ORIGIN
1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
721 agcggccgcc accatgggca agcccatccc taaccccctg ttggggctgg acagcaccgc
781 tcccaaaaag aaaaggaagg tgggcattca cggcgtgcct gcggccgaca aaaagtacag
841 catcggcctt gatatcggca ccaatagcgt gggctgggcc gttatcacag acgaatacaa
901 ggtacccagc aagaagttca aggtgctggg gaatacagac aggcactcta tcaagaaaaa
961 ccttatcggg gctctgctgt ttgactcagg cgagaccgcc gaggccacca ggttgaagag
1021 gaccgcaagg cgaaggtaca cccggaggaa gaacaggatc tgctatctgc aggagatctt
1081 cagcaacgag atggccaagg tggacgacag cttcttccac aggctggagg agagcttcct
1141 tgtcgaggag gataagaagc acgaacgaca ccccatcttc ggcaacatag tcgacgaggt
1201 cgcttatcac gagaagtacc ccaccatcta ccacctgcga aagaaattgg tggatagcac
1261 cgataaagcc gacttgcgac ttatctactt ggctctggcg cacatgatta agttcagggg
1321 ccacttcctg atcgagggcg accttaaccc cgacaacagt gacgtagaca aattgttcat
1381 ccagcttgta cagacctata accagctgtt cgaggaaaac cctattaacg ccagcggggt
1441 ggatgcgaag gccatactta gcgccaggct gagcaaaagc aggcgcttgg agaacctgat
1501 agcccagctg cccggtgaaa agaagaacgg cctcttcggt aatctgattg ccctgagcct
1561 gggcctgacc cccaacttca agagcaactt cgacctggca gaagatgcca agctgcagtt
1621 gagtaaggac acctatgacg acgacttgga caatctgctc gcccaaatcg gcgaccagta
1681 cgctgacctg ttcctcgccg ccaagaacct ttctgacgca atcctgctta gcgatatcct
1741 tagggtgaac acagagatca ccaaggcccc cctgagcgcc agcatgatca agaggtacga
1801 cgagcaccat caggacctga cccttctgaa ggccctggtg aggcagcaac tgcccgagaa
1861 gtacaaggag atctttttcg accagagcaa gaacggctac gccggctaca tcgacggcgg
1921 agccagccaa gaggagttct acaagttcat caagcccatc ctggagaaga tggatggcac
1981 cgaggagctg ctggtgaagc tgaacaggga agatttgctc cggaagcaga ggacctttga
2041 caacggtagc atcccccacc agatccacct gggcgagctg cacgcaatac tgaggcgaca
2101 ggaggatttc taccccttcc tcaaggacaa tagggagaaa atcgaaaaga ttctgacctt
2161 caggatcccc tactacgtgg gccctcttgc caggggcaac agccgattcg cttggatgac
2221 aagaaagagc gaggagacca tcaccccctg gaacttcgag gaagtggtgg acaaaggagc
2281 aagcgcgcag tctttcatcg aacggatgac caatttcgac aaaaacctgc ctaacgagaa
2341 ggtgctgccc aagcacagcc tgctttacga gtacttcacc gtgtacaacg agctcaccaa
2401 ggtgaaatat gtgaccgagg gcatgcgaaa acccgctttc ctgagcggcg agcagaagaa
2461 ggccatcgtg gacctgctgt tcaagaccaa caggaaggtg accgtgaagc agctgaagga
2521 ggactacttc aagaagatcg agtgctttga tagcgtggaa ataagcggcg tggaggacag
2581 gttcaacgcc agcctgggca cctaccacga cttgttgaag ataatcaaag acaaggattt
2641 cctggataat gaggagaacg aggatatact cgaggacatc gtgctgactt tgaccctgtt
2701 tgaggaccga gagatgattg aagaaaggct caaaacctac gcccacctgt tcgacgacaa
2761 agtgatgaaa caactgaaga gacgaagata caccggctgg ggcagactgt ccaggaagct
2821 catcaacggc attagggaca agcagagcgg caagaccatc ctggatttcc tgaagtccga
2881 cggcttcgcc aaccgaaact tcatgcagct gattcacgat gacagcttga ccttcaagga
2941 ggacatccag aaggcccagg ttagcggcca gggcgactcc ctgcacgaac atattgcaaa
3001 cctggcaggc tcccctgcga tcaagaaggg catactgcag accgttaagg ttgtggacga
3061 attggtcaag gtcatgggca ggcacaagcc cgaaaacata gttatagaga tggccagaga
3121 gaaccagacc acccaaaagg gccagaagaa cagccgggag cgcatgaaaa ggatcgagga
3181 gggtatcaag gaactcggaa gccagatcct caaagagcac cccgtggaga atacccagct
3241 ccagaacgag aagctgtacc tgtactacct gcagaacggc agggacatgt acgttgacca
3301 ggagttggac atcaacaggc tttcagacta tgacgtggat cacatagtgc cccagagctt
3361 tcttaaagac gatagcatcg acaacaaggt cctgacccgc tccgacaaaa acaggggcaa
3421 aagcgacaac gtgccaagcg aagaggtggt taaaaagatg aagaactact ggaggcaact
3481 gctcaacgcg aaattgatca cccagagaaa gttcgataac ctgaccaagg ccgagagggg
3541 cggactctcc gaacttgaca aagcgggctt cataaagagg cagctggtcg agacccgaca
3601 gatcacgaag cacgtggccc aaatcctcga cagcagaatg aataccaagt acgatgagaa
3661 tgacaaactc atcagggaag tgaaagtgat taccctgaag agcaagttgg tgtccgactt
3721 tcgcaaagat ttccagttct acaaggtgag ggagatcaac aactaccacc atgcccacga
3781 cgcatacctg aacgccgtgg tcggcaccgc cctgattaag aagtatccaa agctggagtc
3841 cgaatttgtc tacggcgact acaaagttta cgatgtgagg aagatgatcg ctaagagcga
3901 acaggagatc ggcaaggcca ccgctaagta tttcttctac agcaacatca tgaacttttt
3961 caagaccgag atcacacttg ccaacggcga aatcaggaag aggccgctta tcgagaccaa
4021 cggtgagacc ggcgagatcg tgtgggacaa gggcagggac ttcgccaccg tgaggaaagt
4081 cctgagcatg ccccaggtga atattgtgaa aaaaactgag gtgcagacag gcggctttag
4141 caaggaatcc atcctgccca agaggaacag cgacaagctg atcgcccgga agaaggactg
4201 ggaccctaag aagtatggag gcttcgacag ccccaccgta gcctacagcg tgctggtggt
4261 cgcgaaggta gagaagggga agagcaagaa actgaagagc gtgaaggagc tgctcggcat
4321 aaccatcatg gagaggtcca gctttgagaa gaaccccatt gactttttgg aagccaaggg
4381 ctacaaagag gtcaaaaagg acctgatcat caaactcccc aagtactccc tgtttgaatt
4441 ggagaacggc agaaagagga tgctggcgag cgctggggaa ctgcaaaagg gcaacgaact
4501 ggcgctgccc agcaagtacg tgaattttct gtacctggcg tcccactacg aaaagctgaa
4561 aggcagcccc gaggacaacg agcagaagca gctgttcgtg gagcagcaca agcattacct
4621 ggacgagata atcgagcaaa tcagcgagtt cagcaagagg gtgattctgg ccgacgcgaa
4681 cctggataag gtcctcagcg cctacaacaa gcaccgagac aaacccatca gggagcaggc
4741 cgagaatatc atacacctgt tcaccctgac aaatctgggc gcacctgcgg cattcaaata
4801 cttcgatacc accatcgaca ggaaaaggta cactagcact aaggaggtgc tggatgccac
4861 cttgatccac cagtccatta ccggcctgta tgagaccagg atcgacctga gccagcttgg
4921 aggcgactct agggcggacc caaaaaagaa aaggaaggtg gaattctcta gaggcagtgg
4981 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaatgaa
5041 cctgagcaaa aacgtgagcg tgagcgtgta tatgaagggg aacgtcaaca atcatgagtt
5101 tgagtacgac ggggaaggtg gtggtgatcc ttatacaggt aaatattcca tgaagatgac
5161 gctacgtggt caaaattccc tacccttttc ctatgatatc attaccacgg catttcagta
5221 tggtttccgc gtatttacaa aataccctga gggaattgtt gactatttta aggactcgct
5281 tcccgacgca ttccagtgga acagacgaat tgtgtttgaa gatggtggag tactaaacat
5341 gagcagtgat atcacatata aagataatgt tctgcatggt gacgtcaagg ctgagggagt
5401 gaacttcccg ccgaatgggc cagtgatgaa gaatgaaatt gtgatggagg aaccgactga
5461 agaaacattt actccaaaaa acggggttct tgttggcttt tgtcccaaag cgtacttact
5521 taaagacggt tcctattact atggaaatat gacaacattt tacagatcca agaaatctgg
5581 ccaggcacct cctgggtatc actttgttaa gcatcgtctc gtcaagacca atgtgggaca
5641 tggatttaag acggttgagc agactgaata tgccactgct catgtcagtg atcttcccaa
5701 gttcgaagct tgataatgag tttaaacggg ggaggctaac tgaaacacgg aaggagacaa
5761 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacgggtgtt
5821 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
5881 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
5941 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagca
6001 gatctgcgca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
6061 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
6121 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
6181 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
6241 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
6301 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
6361 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
6421 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattaag gtcgggcagg
6481 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
6541 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
6601 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
6661 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
6721 gacgaaacac cgtcaagagg aacaagcttt actcaagagg ttttagagct agaaatagca
6781 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
6841 tctagtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
6901 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
6961 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
7021 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
7081 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
7141 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
7201 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
7261 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
7321 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
7381 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
7441 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
7501 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
7561 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
7621 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
7681 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
7741 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
7801 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
7861 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
7921 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
7981 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
8041 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
8101 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
8161 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
8221 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
8281 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
8341 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
8401 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
8461 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
8521 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
8581 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
8641 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
8701 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
8761 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
8821 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
8881 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
8941 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
9001 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
9061 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
9121 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
9181 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagg
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
| P4900/pCMV-SPORT6-INTS7人源基因质粒 |
| P4901/pCMV-SPORT6-GHDC人源基因质粒 |
| P4902/pCMV-SPORT6-TET3人源基因质粒 |
| P4903/pCMV-SPORT6-ZNF212人源基因质粒 |
| P4904/pCMV-SPORT6-RNF213人源基因质粒 |
| P4905/pCMV-SPORT6-ERCC5人源基因质粒 |
| P4906/pCMV-SPORT6-PIK3R3人源基因质粒 |
| P4907/pCMV-SPORT6-WDR26（850-1986bp）人源基因质粒 |
| P4908/pCMV-SPORT6-MEMO1人源基因质粒 |
| P4909/pCMV-SPORT6-FAM131A人源基因质粒 |
| P4910/pCMV-SPORT6-CA10人源基因质粒 |
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GeneArt CRISPR Nuclease Vector-OFP-gRNA人源基因引导质粒
￥100 - 1000
