| 出品公司: | ZYbscience |
|---|---|
| 载体名称: | pCas9D10A-GFP 或 pCas9D10A_GFP |
| 质粒类型: | CRISPR/Cas9载体；基因敲除载体；哺乳动物细胞载体；双顺反子载体 |
| 高拷贝/低拷贝: | 高拷贝 |
| 插入位点: | 限制性内切酶，多克隆位点 |
| 启动子: | CBh |
| 载体大小: | 9271 bp |
| 5' 测序引物及序列: | ggctctagtgcctctgctaacc |
| 3' 测序引物及序列: | M13 Reverse：CAGGAAACAGCTATGAC |
| 载体标签: | -- |
| 载体抗性: | 氨苄青霉素 |
| 筛选标记: | EGFP绿色荧光蛋白 |
| 克隆菌株: | Stbl3 |
| 宿主细胞（系）: | 哺乳动物细胞 |
| 备注: | -- |
| 产品目录号: | #44720 |
| 稳定性: | 瞬表达或稳表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
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- 详细信息
- 技术资料
- 保存条件：
-20℃低温保存
- 保质期：
三年
- 英文名：
pCas9D10A-GFP
- 库存：
20
- 供应商：
泽叶生物
载体基本信息
载体质粒图谱和多克隆位点信息
载体简介
载体序列
LOCUS Exported File 9271 bp ds-DNA circular SYN 23-七月-2016
DEFINITION Plasmid for co-expression in mammalian cells of enhanced GFP (EGFP)
and human codon-optimized Cas9 with the D10A nickase mutation.
ACCESSION .
VERSION .
KEYWORDS pCas9D10A_GFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 9271)
AUTHORS Musunuru Lab / Addgene #44720
TITLE Direct Submission
JOURNAL Exported 2016-0a7-23 from SnapGene Viewer 1.5.3
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..9271
/organism="synthetic DNA construct"
/lab_host="Mammalian Cells"
/mol_type="other DNA"
enhancer 8..387
/note="CMV enhancer"
/note="human cytomegalovirus immediate early enhancer"
/note="color: #000000"
promoter 390..665
/note="chicken beta-actin promoter"
/note="color: #000000; direction: RIGHT"
intron 666..1678
/note="chimeric intron"
/note="chimera between introns from chicken?beta-actin and
rabbit?beta-globin"
/note="color: #ffffff"
CDS 1745..5848
/codon_start=1
/locus_tag="
"
/product="nickase mutant of the Cas9 endonuclease from
the?Streptococcus pyogenes?Type II CRISPR/Cas system"
/note="Cas9(D10A)"
/note="generates RNA-guided single strand nicks in DNA due
to the D10A mutation in the RuvC catalytic domain"
/note="This feature has 3 segments:
???1:?1745?..?1771?/?#993366
???2:?1772?..?1774?/?#cc99b2
???3:?1775?..?5848?/?#993366"
/protein_id="
"
/translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
DATLIHQSITGLYETRIDLSQLGGD"
CDS 5861..5881
/codon_start=1
/locus_tag="
"
/product="nuclear localization signal of SV40 large T
antigen"
/note="SV40 NLS"
/note="
"
/note="color: #993366"
/protein_id="
"
/translation="PKKKRKV"
CDS 5897..5950
/codon_start=1
/product="2A peptide from?Thosea asigna virus capsid
protein"
/note="T2A"
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/note="color: #cc99b2
Cleavage site after base 5947"
/protein_id="
"
/translation="EGRGSLLTCGDVEENPGP"
CDS 5951..6670
/codon_start=1
/product="enhanced GFP"
/note="EGFP"
/note="mammalian codon-optimized"
/note="This feature has 3 segments:
???1:?5951?..?5953?/?#05fd14
???2:?5954?..?5956?/?#05fd14?/?1a
???3:?5957?..?6670?/?#05fd14"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
EFVTAAGITLGMDELYK"
polyA_signal 6713..6834
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
/note="color: #a6acb3"
rep_origin complement(7285..7873)
/direction=LEFT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
/note="color: #ffff00"
CDS complement(8044..8904)
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/note="AmpR"
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/note="This feature has 2 segments:
???1:?8044?..?8835?/?#ccffcc
???2:?8836?..?8904?/?#ccffcc?/?signal?sequence
Cleavage site after base 8835"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
promoter complement(8905..9009)
/gene="bla"
/note="AmpR promoter"
/note="color: #000000; direction: LEFT"
ORIGIN
1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
181 atagggactt tccattgacg tcaatgggtg gactatttac ggtaaactgc ccacttggca
241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
361 tacgtattag tcatcgctat taccatgggt cgaggtgagc cccacgttct gcttcactct
421 ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg
481 tgcagcgatg ggggcggggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg
541 ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa
601 agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc
661 gggcgggagt cgctgcgttg ccttcgcccc gtgccccgct ccgcgccgcc tcgcgccgcc
721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
781 ctccgggctg taattagcgc ttggtttaat gacggctcgt ttcttttctg tggctgcgtg
841 aaagccttaa agggctccgg gagggccctt tgtgcggggg ggagcggctc ggggggtgcg
901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggcccgc gctgcccggc ggctgtgagc
961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcgtgtg cgcgagggga gcgcggccgg
1021 gggcggtgcc ccgcggtgcg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg
1081 tgcgtggggg ggtgagcagg gggtgtgggc gcggcggtcg ggctgtaacc cccccctgca
1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccat ctccagcctc ggggctgtcc
1561 gcagggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
1621 gaccggcggc tctagtgcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcgcggccgc
1741 caccatggac aagaagtact ccattgggct cgctatcggc acaaacagcg tcggctgggc
1801 cgtcattacg gacgagtaca aggtgccgag caaaaaattc aaagttctgg gcaataccga
1861 tcgccacagc ataaagaaga acctcattgg cgccctcctg ttcgactccg gggagacggc
1921 cgaagccacg cggctcaaaa gaacagcacg gcgcagatat acccgcagaa agaatcggat
1981 ctgctacctg caggagatct ttagtaatga gatggctaag gtggatgact ctttcttcca
2041 taggctggag gagtcctttt tggtggagga ggataaaaag cacgagcgcc acccaatctt
2101 tggcaatatc gtggacgagg tggcgtacca tgaaaagtac ccaaccatat atcatctgag
2161 gaagaagctt gtagacagta ctgataaggc tgacttgcgg ttgatctatc tcgcgctggc
2221 gcatatgatc aaatttcggg gacacttcct catcgagggg gacctgaacc cagacaacag
2281 cgatgtcgac aaactcttta tccaactggt tcagacttac aatcagcttt tcgaagagaa
2341 cccgatcaac gcatccggag ttgacgccaa agcaatcctg agcgctaggc tgtccaaatc
2401 ccggcggctc gaaaacctca tcgcacagct ccctggggag aagaagaacg gcctgtttgg
2461 taatcttatc gccctgtcac tcgggctgac ccccaacttt aaatctaact tcgacctggc
2521 cgaagatgcc aagcttcaac tgagcaaaga cacctacgat gatgatctcg acaatctgct
2581 ggcccagatc ggcgaccagt acgcagacct ttttttggcg gcaaagaacc tgtcagacgc
2641 cattctgctg agtgatattc tgcgagtgaa cacggagatc accaaagctc cgctgagcgc
2701 tagtatgatc aagcgctatg atgagcacca ccaagacttg actttgctga aggcccttgt
2761 cagacagcaa ctgcctgaga agtacaagga aattttcttc gatcagtcta aaaatggcta
2821 cgccggatac attgacggcg gagcaagcca ggaggaattt tacaaattta ttaagcccat
2881 cttggaaaaa atggacggca ccgaggagct gctggtaaag cttaacagag aagatctgtt
2941 gcgcaaacag cgcactttcg acaatggaag catcccccac cagattcacc tgggcgaact
3001 gcacgctatc ctcaggcggc aagaggattt ctaccccttt ttgaaagata acagggaaaa
3061 gattgagaaa atcctcacat ttcggatacc ctactatgta ggccccctcg cccggggaaa
3121 ttccagattc gcgtggatga ctcgcaaatc agaagagacc atcactccct ggaacttcga
3181 ggaagtcgtg gataaggggg cctctgccca gtccttcatc gaaaggatga ctaactttga
3241 taaaaatctg cctaacgaaa aggtgcttcc taaacactct ctgctgtacg agtacttcac
3301 agtttataac gagctcacca aggtcaaata cgtcacagaa gggatgagaa agccagcatt
3361 cctgtctgga gagcagaaga aagctatcgt ggacctcctc ttcaagacga accggaaagt
3421 taccgtgaaa cagctcaaag aagactattt caaaaagatt gaatgtttcg actctgttga
3481 aatcagcgga gtggaggatc gcttcaacgc atccctggga acgtatcacg atctcctgaa
3541 aatcattaaa gacaaggact tcctggacaa tgaggagaac gaggacattc ttgaggacat
3601 tgtcctcacc cttacgttgt ttgaagatag ggagatgatt gaagaacgct tgaaaactta
3661 cgctcatctc ttcgacgaca aagtcatgaa acagctcaag aggcgccgat atacaggatg
3721 ggggcggctg tcaagaaaac tgatcaatgg gatccgagac aagcagagtg gaaagacaat
3781 cctggatttt cttaagtccg atggatttgc caaccggaac ttcatgcagt tgatccatga
3841 tgactctctc acctttaagg aggacatcca gaaagcacaa gtttctggcc agggggacag
3901 tcttcacgag cacatcgcta atcttgcagg tagcccagct atcaaaaagg gaatactgca
3961 gaccgttaag gtcgtggatg aactcgtcaa agtaatggga aggcataagc ccgagaatat
4021 cgttatcgag atggcccgag agaaccaaac tacccagaag ggacagaaga acagtaggga
4081 aaggatgaag aggattgaag agggtataaa agaactgggg tcccaaatcc ttaaggaaca
4141 cccagttgaa aacacccagc ttcagaatga gaagctctac ctgtactacc tgcagaacgg
4201 cagggacatg tacgtggatc aggaactgga catcaatcgg ctctccgact acgacgtgga
4261 tcatatcgtg ccccagtctt ttctcaaaga tgattctatt gataataaag tgttgacaag
4321 atccgataaa aatagaggga agagtgataa cgtcccctca gaagaagttg tcaagaaaat
4381 gaaaaattat tggcggcagc tgctgaacgc caaactgatc acacaacgga agttcgataa
4441 tctgactaag gctgaacgag gtggcctgtc tgagttggat aaagccggct tcatcaaaag
4501 gcagcttgtt gagacacgcc agatcaccaa gcacgtggcc caaattctcg attcacgcat
4561 gaacaccaag tacgatgaaa atgacaaact gattcgagag gtgaaagtta ttactctgaa
4621 gtctaagctg gtctcagatt tcagaaagga ctttcagttt tataaggtga gagagatcaa
4681 caattaccac catgcgcatg atgcctacct gaatgcagtg gtaggcactg cacttatcaa
4741 aaaatatccc aagcttgaat ctgaatttgt ttacggagac tataaagtgt acgatgttag
4801 gaaaatgatc gcaaagtctg agcaggaaat aggcaaggcc accgctaagt acttctttta
4861 cagcaatatt atgaattttt tcaagaccga gattacactg gccaatggag agattcggaa
4921 gcgaccactt atcgaaacaa acggagaaac aggagaaatc gtgtgggaca agggtaggga
4981 tttcgcgaca gtccggaagg tcctgtccat gccgcaggtg aacatcgtta aaaagaccga
5041 agtacagacc ggaggcttct ccaaggaaag tatcctcccg aaaaggaaca gcgacaagct
5101 gatcgcacgc aaaaaagatt gggaccccaa gaaatacggc ggattcgatt ctcctacagt
5161 cgcttacagt gtactggttg tggccaaagt ggagaaaggg aagtctaaaa aactcaaaag
5221 cgtcaaggaa ctgctgggca tcacaatcat ggagcgatca agcttcgaaa aaaaccccat
5281 cgactttctc gaggcgaaag gatataaaga ggtcaaaaaa gacctcatca ttaagcttcc
5341 caagtactct ctctttgagc ttgaaaacgg ccggaaacga atgctcgcta gtgcgggcga
5401 gctgcagaaa ggtaacgagc tggcactgcc ctctaaatac gttaatttct tgtatctggc
5461 cagccactat gaaaagctca aagggtctcc cgaagataat gagcagaagc agctgttcgt
5521 ggaacaacac aaacactacc ttgatgagat catcgagcaa ataagcgaat tctccaaaag
5581 agtgatcctc gccgacgcta acctcgataa ggtgctttct gcttacaata agcacaggga
5641 taagcccatc agggagcagg cagaaaacat tatccacttg tttactctga ccaacttggg
5701 cgcgcctgca gccttcaagt acttcgacac caccatagac agaaagcggt acacctctac
5761 aaaggaggtc ctggacgcca cactgattca tcagtcaatt acggggctct atgaaacaag
5821 aatcgacctc tctcagctcg gtggagacag cagggctgac cccaagaaga agaggaaggt
5881 gaggtccggc ggcggagagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa
5941 tcccggccca atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
6001 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
6061 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
6121 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
6181 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
6241 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
6301 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
6361 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
6421 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
6481 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
6541 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
6601 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
6661 gtacaagtaa ctgcagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt
6721 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
6781 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
6841 ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
6901 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
6961 agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
7021 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
7081 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
7141 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
7201 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
7261 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
7321 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
7381 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
7441 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
7501 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
7561 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
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pCas9D10A-GFP载体
¥1000
