| 質粒名稱: | pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry |
|---|---|
| 出品公司: | Addgene |
| 目錄編號: | 35512 |
| 存儲實驗室: | Karl Deisseroth |
|
載體骨架基本信息 |
|
| 載體名稱: | pAAV |
| 載體抗性: | 氨芐青霉素 |
| 空載體大小: | 5385 bp |
| 載體修飾: | 添加CaMKIIa促進劑和WPRE |
| 載體類型: | 哺乳細胞表達,AAV |
| 高拷貝/低拷貝: | 高拷貝 |
| 生長條件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名稱: | hChR2 |
| 物種來源: | 萊茵衣藻 |
| 插入大小: | 1646 bp |
| 基因突變: | E123T and T159C |
| 啟動子 | CaMKIIa |
| 標簽/融合蛋白: | mCherry（插入件上的C端子） |
| 克隆方法: | 限制性內切酶 |
| 5' 克隆位點: | BamHI(未破壞) |
| 3' 克隆位點: | EcoRI(未破壞) |
| 5' 測序引物 | AGTCCTGCAGTATTGTGTAT |
| 3' 測序引物 | GCAATAGCATGATACAAAGG |
|
文獻引用方法 |
|
| 材料和方法部分: | pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry was a gift from Karl Deisseroth (Addgene plasmid # 35512 ; //n2t.net/addgene:35512 ; RRID:Addgene_35512) |
| 參考文獻部分: | 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 |
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pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry載體質粒基本信息
pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry質粒圖譜載體圖譜和pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry載體序列質粒序列多克隆位點信息
-mCherry質粒圖譜.png)
pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry質粒載體簡介
pAAV-CaMKIIa-hChR2(E123T/T159C)-mCherry質粒序列載體序列
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
//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
//