| 质粒名称: | pDCC5 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 59984 |
| 存储实验室: | Peter Duchek |
|
载体骨架基本信息 |
|
| 载体名称: | pHW |
| 载体出品公司: | -- |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | -- |
| 完整质粒大小: | 8088 bp |
| 载体修饰: | -- |
| 载体类型: | 昆虫表达，CRISPR |
| 筛选标记: | -- |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | Cas9-D10A,HA-hSpCas9-D10A |
| 物种来源: | Synthetic |
| 插入大小: | 4209 bp |
| 基因突变: | D10A |
| GenBank编号 | -- |
| 启动子 | hsp70Bb |
| 标签/融合蛋白: | HA（插入N端子） |
| 克隆方法: | Gateway克隆 |
| 5' 克隆位点: | -- |
| 3' 克隆位点: | -- |
| 5' 测序引物 | GCGCTTCCGGAGGTATACACC |
| 3' 测序引物 | -- |
|
文献引用方法 |
|
| 材料和方法部分: | pDCC5 was a gift from Peter Duchek (Addgene plasmid # 59984 ; http://n2t.net/addgene:59984 ; RRID:Addgene_59984) |
| 参考文献部分: | Efficient CRISPR/Cas9 Plasmids for Rapid and Versatile Genome Editing in Drosophila. Gokcezade J, Sienski G, Duchek P. G3 (Bethesda). 2014 Sep 17. pii: g3.114.014126. doi: 10.1534/g3.114.014126. 10.1534/g3.114.014126 PubMed 25236734 |
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载体基本信息
载体图谱质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS pDCC5 8120 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Bi-cistronic Drosophila CRISPR/Cas9 vector, contains hsp70>Cas9-D10A
nickase and U6>sgRNA cassette.
ACCESSION .
VERSION .
KEYWORDS pDCC5
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8120)
AUTHORS Gokcezade J, Sienski G, Duchek P
TITLE Efficient CRISPR/Cas9 Plasmids for Rapid and Versatile Genome
Editing in Drosophila.
JOURNAL G3 (Bethesda). 2014 Sep 17. pii: g3.114.014126. doi:
10.1534/g3.114.014126.
PUBMED 25236734
REFERENCE 2 (bases 1 to 8120)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8120
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 84..483
/label=dU6-2 promoter
/note="RNA polymerase III promoter for Drosophila U6-2
snRNA (Das et al., 1987)"
misc_RNA 502..577
/label=gRNA scaffold
/note="guide RNA scaffold for the Streptococcus pyogenes
CRISPR/Cas9 system"
protein_bind 1101..1125
/gene="mutant version of attB"
/label=attB1
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
CDS 1131..1157
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 1161..1181
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1191..5291
/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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAKNIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5295..5315
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
protein_bind complement(5332..5356)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
intron 5448..5513
/label=small t intron
/note="SV40 (simian virus 40) small t antigen intron"
CDS 5643..5663
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
polyA_signal 6088..6222
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(6125..6144)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 6179..6198
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
CDS 6319..7179
/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 complement(6537..6556)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 7350..7938
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7839..7858
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8092..8109
/label=L4440
/note="L4440 vector, forward primer"
ORIGIN
1 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcaggcaact
61 cgtgaaaggt aggcggatca gcggttcgac ttgcagcctg aaatacggca cgagtaggaa
121 aagccgagtc aaatgccgaa tgcagagtct cattacagca caatcaactc aagaaaaact
181 cgacactttt ttaccatttg cacttaaatc cttttttatt cgttatgtat actttttttg
241 gtccctaacc aaaacaaaac caaactctct tagtcgtgcc tctatattta aaactatcaa
301 tttattatag tcaataaatc gaactgtgtt ttcaacaaac gaacaatagg acactttgat
361 tctaaaggaa attttgaaaa tcttaagcag agggttctta agaccatttg ccaattctta
421 taattctcaa ctgctctttc ctgatgttga tcatttatat aggtatgttt tcctcaatac
481 ttcgggtctt cgagaagacc tgttttagag ctagaaatag caagttaaaa taaggctagt
541 ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag agctagaaat
601 agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc agacaaatgg
661 ctctagaatc ccaaaacaaa ctggttattg tggtaggtca tttgtttggc agaaagaaaa
721 ctcgagaaat ttctctggcc gttattcgtt attctctctt ttctttttgg gtctctccct
781 ctctgcacta atgctctctc actctgtcac acagtaaacg gcatactgct ctcgttggtt
841 cgagagagcg cgcctcgaat gttcgcgaaa agagcgccgg agtataaata gaggcgcttc
901 gtctacggag cgacaattca attcaaacaa gcaaagtgaa cacgtcgcta agcgaaagct
961 aagcaaataa acaagcgcag ctgaacaagc taaacaatct gcagccaagc tcgataagct
1021 tgttcgaatc tcgagtgcgc gcttccggag gtatacacct aggcggtacc actgcagtga
1081 attcggagct ctaccggtat acaagtttgt acaaaaaagc aggctcaatg tacccatacg
1141 atgttccaga ttacgcttcg ccgaagaaaa agcgcaaggt cgaagcgtcc gacaagaagt
1201 acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
1261 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
1321 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
1381 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
1441 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
1501 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
1561 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
1621 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
1681 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
1741 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
1801 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
1861 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg attgccctga
1921 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
1981 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
2041 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
2101 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
2161 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
2221 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
2281 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
2341 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
2401 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
2461 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
2521 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
2581 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
2641 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
2701 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
2761 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
2821 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
2881 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
2941 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
3001 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
3061 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
3121 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
3181 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
3241 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
3301 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
3361 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
3421 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
3481 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
3541 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
3601 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
3661 accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga
3721 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
3781 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
3841 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
3901 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
3961 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
4021 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
4081 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
4141 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
4201 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
4261 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
4321 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
4381 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
4441 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
4501 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
4561 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
4621 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
4681 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
4741 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
4801 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
4861 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
4921 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
4981 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
5041 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
5101 aggccaagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
5161 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
5221 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
5281 tgggaggcga cagccccaag aagaagagaa aggtggaggc cagctaatag gacccagctt
5341 tcttgtacaa agtggtgacg taagctagca ggatctttgt gaaggaacct tacttctgtg
5401 gtgtgacata attggacaaa ctacctacag agatttaaag ctctaaggta aatataaaat
5461 ttttaagtgt ataatgtgtt aaactactga ttctaattgt ttgtgtattt tagattccaa
5521 cctatggaac tgatgaatgg gagcagtggt ggaatgcctt taatgaggaa aacctgtttt
5581 gctcagaaga aatgccatct agtgatgatg aggctactgc tgactctcaa cattctactc
5641 ctccaaaaaa gaagagaaag gtagttgacc ccaaggactt tccttcagaa ttgctaagtt
5701 ttttgagtca tgctgtgttt agtaatagaa ctcttgcttg ctttgctatt tacaccacaa
5761 aggaaaaagc tgcactgcta tacaagaaaa ttatggaaaa atattctgta acctttataa
5821 gtaggcataa cagttataat cataacatac tgttttttct tactccacac aggcatagag
5881 tgtctgctat taataactat gctcaaaaat tgtgtacctt tagcttttta atttgtaaag
5941 gggttaataa ggaatatttg atgtatagtg ccttgactag agatcataat cagccatacc
6001 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa
6061 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
6121 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
6181 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcccgtttaa actacgcgta
6241 attcaaacag ggttctggcg tcgttctcgt actgttttcc ccaggccagt gctttagcgt
6301 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
6361 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
6421 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
6481 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
6541 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
6601 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
6661 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
6721 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
6781 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
6841 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
6901 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
6961 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
7021 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
7081 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
7141 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
7201 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
7261 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
7321 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
7381 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
7441 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
7501 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
7561 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
7621 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
7681 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
7741 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
7801 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
7861 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
7921 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
7981 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
8041 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
8101 gtgagcgagg aagcggaaga
//
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