| 质粒名称: | pDCC6 |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 59985 |
| 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: | 8122 bp |
| 完整质粒大小: | -- |
| 载体修饰: | -- |
| 载体类型: | 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: | Cas9,3xFLAG-hSpCas9 |
| 物种来源: | Synthetic; S. pyogenes |
| 插入大小: | 4266 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | hsp70Bb |
| 标签/融合蛋白: | 3xFLAG（插入件上的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: | pDCC6 was a gift from Peter Duchek (Addgene plasmid # 59985 ; http://n2t.net/addgene:59985 ; RRID:Addgene_59985) |
| 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 |
买家导航
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 pDCC6 8154 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Bi-cistronic Drosophila CRISPR/Cas9 vector, contains hsp70>Cas9 and
U6>sgRNA cassette.
ACCESSION .
VERSION .
KEYWORDS pDCC6
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 8154)
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 8154)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..8154
/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"
regulatory 1099..1108
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1108..1173
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
CDS 1180..1200
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1225..5325
/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
GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
NLDKVLSAYNKHRDKPIREQAKNIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQSITGLYETRIDLSQLGGD"
CDS 5329..5349
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
protein_bind complement(5366..5390)
/gene="mutant version of attB"
/label=attB2
/bound_moiety="BP Clonase(TM)"
/note="recombination site for the Gateway(R) BP reaction"
intron 5482..5547
/label=small t intron
/note="SV40 (simian virus 40) small t antigen intron"
CDS 5677..5697
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
polyA_signal 6122..6256
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(6159..6178)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 6213..6232
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
CDS 6353..7213
/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(6571..6590)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 7384..7972
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7873..7892
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 8126..8143
/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 ctaccggtgc caccatggac tataaggacc acgacggaga ctacaaggat
1141 catgatattg attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc
1201 ggtatccacg gagtcccagc agccgacaag aagtacagca tcggcctgga catcggcacc
1261 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
1321 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
1381 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
1441 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
1501 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
1561 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
1621 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
1681 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
1741 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
1801 cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc catcctgtct
1861 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
1921 aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
1981 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
2041 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
2101 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
2161 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
2221 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
2281 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
2341 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
2401 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
2461 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
2521 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
2581 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
2641 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
2701 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
2761 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
2821 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
2881 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
2941 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
3001 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
3061 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
3121 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
3181 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
3241 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
3301 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
3361 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
3421 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
3481 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
3541 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
3601 cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa gctgtacctg
3661 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
3721 tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga ctccatcgac
3781 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
3841 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
3901 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
3961 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
4021 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
4081 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
4141 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
4201 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
4261 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
4321 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
4381 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
4441 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
4501 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
4561 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
4621 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
4681 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
4741 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
4801 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
4861 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
4921 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
4981 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
5041 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
5101 tacaacaagc accgggataa gcccatcaga gagcaggcca agaatatcat ccacctgttt
5161 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
5221 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
5281 ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacagccc caagaagaag
5341 agaaaggtgg aggccagcta ataggaccca gctttcttgt acaaagtggt gacgtaagct
5401 agcaggatct ttgtgaagga accttacttc tgtggtgtga cataattgga caaactacct
5461 acagagattt aaagctctaa ggtaaatata aaatttttaa gtgtataatg tgttaaacta
5521 ctgattctaa ttgtttgtgt attttagatt ccaacctatg gaactgatga atgggagcag
5581 tggtggaatg cctttaatga ggaaaacctg ttttgctcag aagaaatgcc atctagtgat
5641 gatgaggcta ctgctgactc tcaacattct actcctccaa aaaagaagag aaaggtagtt
5701 gaccccaagg actttccttc agaattgcta agttttttga gtcatgctgt gtttagtaat
5761 agaactcttg cttgctttgc tatttacacc acaaaggaaa aagctgcact gctatacaag
5821 aaaattatgg aaaaatattc tgtaaccttt ataagtaggc ataacagtta taatcataac
5881 atactgtttt ttcttactcc acacaggcat agagtgtctg ctattaataa ctatgctcaa
5941 aaattgtgta cctttagctt tttaatttgt aaaggggtta ataaggaata tttgatgtat
6001 agtgccttga ctagagatca taatcagcca taccacattt gtagaggttt tacttgcttt
6061 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
6121 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
6181 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
6241 ttatcatgtc tggatcccgt ttaaactacg cgtaattcaa acagggttct ggcgtcgttc
6301 tcgtactgtt ttccccaggc cagtgcttta gcgttattga aaaaggaaga gtatgagtat
6361 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
6421 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
6481 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
6541 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
6601 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
6661 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
6721 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
6781 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
6841 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
6901 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
6961 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
7021 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
7081 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
7141 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
7201 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
7261 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
7321 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
7381 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
7441 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
7501 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
7561 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
7621 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
7681 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
7741 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
7801 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
7861 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
7921 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
7981 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
8041 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
8101 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aaga
//
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