质粒名称: | pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry |
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出品公司: | Addgene |
目录编号: | 35512 |
存储实验室: | Karl Deisseroth |
载体骨架基本信息 |
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载体名称: | pAAV |
载体抗性: | 氨苄青霉素 |
空载体大小: | 5385 bp |
载体修饰: | 添加CaMKIIa促进剂和WPRE |
载体类型: | 哺乳细胞表达,AAV |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | hChR2 |
物种来源: | 莱茵衣藻 |
插入大小: | 1646 bp |
基因突变: | E123T and T159C |
启动子 | CaMKIIa |
标签/融合蛋白: | mCherry(插入件上的C端子) |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | BamHI(未破坏) |
3' 克隆位点: | EcoRI(未破坏) |
5' 测序引物 | AGTCCTGCAGTATTGTGTAT |
3' 测序引物 | GCAATAGCATGATACAAAGG |
文献引用方法 |
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材料和方法部分: | pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry was a gift from Karl Deisseroth (Addgene plasmid # 35512 ; http://n2t.net/addgene:35512 ; RRID:Addgene_35512) |
参考文献部分: | Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R, Gunaydin LA, Hyun M, Fenno LE, Gradinaru V, Yizhar O, Deisseroth K. Nat Methods. 2011 Dec 18;9(2):159-72. doi: 10.1038/nmeth.1808. 10.1038/nmeth.1808 PubMed 22179551 |
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 pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry 7020 bp ds-DNA circular SYN 06-AUG-2018 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7020) AUTHORS Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O'Shea DJ, Prakash R, Gunaydin LA, Hyun M, Fenno LE, Gradinaru V, Yizhar O, Deisseroth K TITLE Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. JOURNAL Nat Methods. 2011 Dec 18;9(2):159-72. doi: 10.1038/nmeth.1808. PUBMED 22179551 REFERENCE 2 (bases 1 to 7020) AUTHORS . TITLE Direct Submission JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7020 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 52..181 /label=AAV2 ITR (alternate) /note="Functional equivalent of wild-type AAV2 ITR" regulatory 1521..1530 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2466..3173 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(2608..2626) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 2876..2895 /label=mCherry-F /note="mCherry, forward primer" misc_feature 3198..3786 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(3251..3271) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(3669..3680) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" polyA_signal 3818..4294 /label=hGH poly(A) signal /note="human growth hormone polyadenylation signal" primer_bind complement(4017..4036) /label=hGH-PA-R /note="Human growth hormone terminator, reverse primer" repeat_region 4334..4474 /label=AAV2 ITR /note="inverted terminal repeat of adeno-associated virus serotype 2" repeat_region 4334..4463 /label=AAV2 ITR rep_origin 4549..5004 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4636..4655) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4846..4867 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(5021..5040) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 5140..5162 /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind complement(5200..5218) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 5286..5390 /gene="bla" /label=AmpR promoter CDS 5391..6251 /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(5609..5628) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 6422..7010 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 6911..6930 /label=pBR322ori-F /note="pBR322 origin, forward primer" ORIGIN 1 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg tcctgcaggc 61 agctgcgcgc tcgctcgctc actgaggccg cccgggcgtc gggcgacctt tggtcgcccg 121 gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc 181 tgcggccgca cgcgtttaac attatggcct taggtcactt catctccatg gggttcttct 241 tctgattttc tagaaaatga gatgggggtg cagagagctt cctcagtgac ctgcccaggg 301 tcacatcaga aatgtcagag ctagaacttg aactcagatt actaatctta aattccatgc 361 cttgggggca tgcaagtacg atatacagaa ggagtgaact cattagggca gatgaccaat 421 gagtttagga aagaagagtc cagggcaggg tacatctaca ccacccgccc agccctgggt 481 gagtccagcc acgttcacct cattatagtt gcctctctcc agtcctacct tgacgggaag 541 cacaagcaga aactgggaca ggagccccag gagaccaaat cttcatggtc cctctgggag 601 gatgggtggg gagagctgtg gcagaggcct caggaggggc cctgctgctc agtggtgaca 661 gataggggtg agaaagcaga cagagtcatt ccgtcagcat tctgggtctg tttggtactt 721 cttctcacgc taaggtggcg gtgtgatatg cacaatggct aaaaagcagg gagagctgga 781 aagaaacaag gacagagaca gaggccaagt caaccagacc aattcccaga ggaagcaaag 841 aaaccattac agagactaca agggggaagg gaaggagaga tgaattagct tcccctgtaa 901 accttagaac ccagctgttg ccagggcaac ggggcaatac ctgtctcttc agaggagatg 961 aagttgccag ggtaactaca tcctgtcttt ctcaaggacc atcccagaat gtggcaccca 1021 ctagccgtta ccatagcaac tgcctctttg ccccacttaa tcccatcccg tctgttaaaa 1081 gggccctata gttggaggtg ggggaggtag gaagagcgat gatcacttgt ggactaagtt 1141 tgttcgcatc cccttctcca accccctcag tacatcaccc tgggggaaca gggtccactt 1201 gctcctgggc ccacacagtc ctgcagtatt gtgtatataa ggccagggca aagaggagca 1261 ggttttaaag tgaaaggcag gcaggtgttg gggaggcagt taccggggca acgggaacag 1321 ggcgtttcgg aggtggttgc catggggacc tggatgctga cgaaggctcg cgaggctgtg 1381 agcagccaca gtgccctgct cagaagcccc aagctcgtca gtcaagccgg ttctccgttt 1441 gcactcagga gcacgggcag gcgagtggcc cctagttctg ggggcagctc tagagcggta 1501 ccggatcccc gggtaccggt gccaccatgg actatggcgg cgctttgtct gccgtcggac 1561 gcgaactttt gttcgttact aatcctgtgg tggtgaacgg gtccgtcctg gtccctgagg 1621 atcaatgtta ctgtgccgga tggattgaat ctcgcggcac gaacggcgct cagaccgcgt 1681 caaatgtcct gcagtggctt gcagcaggat tcagcatttt gctgctgatg ttctatgcct 1741 accaaacctg gaaatctaca tgcggctggg aggagatcta tgtgtgcgcc attgaaatgg 1801 ttaaggtgat tctcgagttc ttttttgagt ttaagaatcc ctctatgctc taccttgcca 1861 caggacaccg ggtgcagtgg ctgcgctatg caacctggct gctcacttgt cctgtcatcc 1921 ttatccacct gagcaacctc accggcctga gcaacgacta cagcaggaga accatgggac 1981 tccttgtctc agacatcggg tgtatcgtgt ggggggctac cagcgccatg gcaaccggct 2041 atgttaaagt catcttcttt tgtcttggat tgtgctatgg cgcgaacaca ttttttcacg 2101 ccgccaaagc atatatcgag ggttatcata ctgtgccaaa gggtcggtgc cgccaggtcg 2161 tgaccggcat ggcatggctg tttttcgtga gctggggtat gttcccaatt ctcttcattt 2221 tggggcccga aggttttggc gtcctgagcg tctatggctc caccgtaggt cacacgatta 2281 ttgatctgat gagtaaaaat tgttgggggt tgttgggaca ctacctgcgc gtcctgatcc 2341 acgagcacat attgattcac ggagatatcc gcaaaaccac caaactgaac atcggcggaa 2401 cggagatcga ggtcgagact ctcgtcgaag acgaagccga ggccggagcc gtgccagcgg 2461 ccgccgtgag caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca 2521 aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg 2581 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc 2641 ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc 2701 accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc 2761 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg 2821 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg 2881 taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg 2941 gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg 3001 ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg 3061 tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg 3121 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taagaattcg 3181 atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta 3241 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc 3301 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt 3361 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg 3421 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccggaactt 3481 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct 3541 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt 3601 cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct 3661 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc 3721 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct 3781 ccccgcatcg ataccgagcg ctgctcgaga gatctacggg tggcatccct gtgacccctc 3841 cccagtgcct ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat 3901 aaaattaagt tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag 3961 gggggtggta tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta 4021 ttgggaacca agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg 4081 ggttcaagcg attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac 4141 caggctcagc taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct 4201 ggtctccaac tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt 4261 acaggcgtga accactgctc ccttccctgt ccttctgatt ttgtaggtaa ccacgtgcgg 4321 accgagcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 4381 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 4441 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt 4501 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta 4561 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca 4621 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 4681 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc 4741 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat 4801 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc 4861 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc 4921 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta 4981 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg 5041 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 5101 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 5161 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt 5221 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa 5281 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 5341 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 5401 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 5461 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 5521 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 5581 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 5641 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 5701 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 5761 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 5821 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 5881 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 5941 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 6001 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 6061 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 6121 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 6181 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 6241 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc 6301 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc 6361 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt 6421 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac 6481 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct 6541 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact 6601 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg 6661 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata 6721 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga 6781 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag 6841 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg 6901 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac 6961 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca //