| 质粒名称: | pAAV_hsyn_NES-his-CaMPARI2-F391W-G395D-WPRE-SV40 |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 101063 |
| 存储实验室: | Eric Schreiter |
|
载体骨架基本信息 |
|
| 载体名称: | pAAV |
| 空载体大小: | 4267bp |
| 完整质粒大小: | 5567 bp |
| 载体类型: | AAV |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | CaMPARI2_F391W-G395D |
| 物种来源: | Synthetic |
| 插入大小: | 1446 bp |
| 启动子: | hsyn (synapsin-1) |
| 标签/融合蛋白: |
NES_his (N terminal on insert)
FLAG-HA-myc (C terminal on insert)
|
| 克隆方法: | Gibson Cloning |
| 5’测序引物 | primerf.hsyn |
| 3’测序引物 | primerr.WPRE |
|
文献引用方法 |
|
| 材料和方法部分: | pAAV_hsyn_NES-his-CaMPARI2-F391W-G395D-WPRE-SV40 was a gift from Eric Schreiter (Addgene plasmid # 101063 ; http://n2t.net/addgene:101063 ; RRID:Addgene_101063) |
| 参考文献部分: | Improved methods for marking active neuron populations. Moeyaert B, Holt G, Madangopal R, Perez-Alvarez A, Fearey BC, Trojanowski NF, Ledderose J, Zolnik TA, Das A, Patel D, Brown TA, Sachdev RNS, Eickholt BJ, Larkum ME, Turrigiano GG, Dana H, Gee CE, Oertner TG, Hope BT, Schreiter ER. Nat Commun. 2018 Oct 25;9(1):4440. doi: 10.1038/s41467-018-06935-2. 10.1038/s41467-018-06935-2 PubMed 30361563 |
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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 pAAV_hsyn_NES-his-CaMPARI2-F391W-G395D-WPRE-SV40 5567 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION AAV vector expressing CaMPARI2_F391W-G395D (Kd = 530nM), a
photoconvertible fluorescent protein-based calcium integrator, in
neurons.
ACCESSION .
VERSION .
KEYWORDS pAAV_hsyn_NES-his-CaMPARI2-F391W-G395D-WPRE-SV40
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5567)
AUTHORS Moeyaert B, Holt G, Madangopal R, Perez-Alvarez A, Fearey BC,
Trojanowski NF, Ledderose J, Zolnik TA, Das A, Patel D, Brown TA,
Sachdev RNS, Eickholt BJ, Larkum ME, Turrigiano GG, Dana H, Gee CE,
Oertner TG, Hope BT, Schreiter ER
TITLE Improved methods for marking active neuron populations.
JOURNAL Nat Commun. 2018 Oct 25;9(1):4440. doi: 10.1038/s41467-018-06935-2.
PUBMED 30361563
REFERENCE 2 (bases 1 to 5567)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5567
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 20..467
/label=hSyn promoter
/note="human synapsin I promoter; confers neuron-specific
expression (Kugler et al., 2003)"
CDS 564..581
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 588..1028
/codon_start=1
/product="calcium-binding protein"
/label=calmodulin
/translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAEL
RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVVMMTAK"
CDS 1824..1847
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 1866..1892
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 1911..1940
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
misc_feature 1962..2550
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(2015..2035)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(2433..2444)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(2553..2569)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2554..2570)
/label=pBluescriptKS
/note="For pBluescript vector"
polyA_signal 2599..2720
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2610..2629)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 2664..2683
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(2928..2945)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(3099..3687)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(3179..3198)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(3858..4718)
/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 4481..4500
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(4719..4823)
/gene="bla"
/label=AmpR promoter
rep_origin complement(4849..5304)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4986..5007)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 5198..5217
/label=F1ori-R
/note="F1 origin, reverse primer"
repeat_region 5376..5505
/label=AAV2 ITR
ORIGIN
1 gcagagggcc ctgcgtatga gtgcaagtgg gttttaggac caggatgagg cggggtgggg
61 gtgcctacct gacgaccgac cccgacccac tggacaagca cccaaccccc attccccaaa
121 ttgcgcatcc cctatcagag agggggaggg gaaacaggat gcggcgaggc gcgtgcgcac
181 tgccagcttc agcaccgcgg acagtgcctt cgcccccgcc tggcggcgcg cgccaccgcc
241 gcctcagcac tgaaggcgcg ctgacgtcac tcgccggtcc cccgcaaact ccccttcccg
301 gccaccttgg tcgcgtccgc gccgccgccg gcccagccgg accgcaccac gcgaggcgcg
361 agataggggg gcacgggcgc gaccatctgc gctgcggcgc cggcgactca gcgctgcctc
421 agtctgcggt gggcagcgga ggagtcgtgt cgtgcctgag agcgcagtcg aattcaagct
481 gctagcaagg atccgccacc atgctgcaga acgagcttgc tcttaagttg gctggacttg
541 atattaacaa gactggaggt tctcatcatc accaccacca tggatccgac caactgactg
601 aagagcagat cgcagaattt aaagaggctt tctccctatt tgacaaggac ggggatggga
661 caataacaac caaggagctg gggacggtga tgcggtctct ggggcagaac cccacagaag
721 cagagctgca ggacatgatc aatgaagtag atgccgacgg tgacggcaca atcgacttcc
781 ctgagttcct gacaatgatg gcaagaaaaa tgaaagacac agacagtgaa gaagaaatta
841 gagaagcgtt ccgtgtgttt gataaggatg gcaatggcta catcagtgca gcagagcttc
901 gccacgtgat gacaaacctt ggagagaagt taacagatga agaggttgat gaaatgatca
961 gggaagcaga catcgatggg gatggtcagg taaactacga agagtttgta gtgatgatga
1021 cagcgaagct cgagtgcgag aaaatctatg tgcgtgatgg agtgctgacg ggtgatattc
1081 atatgatctt gttgcttgaa ggaaatgccc attaccgatg tgacttcaga actacttaca
1141 aagctaagga gaagggtgtc aagttaccag gcgtgcacta tgtggaccac accattgaga
1201 ttttaagcca tgacaaagat tacaacaagg ttaagatcta tgagtatgct gttgctcatt
1261 ctggattgcc tgacaatgcc agacgaggcg gtaccggcgg atccatggtg agtgcgatta
1321 agccagacat gaagatcaaa ctgcgtatgg aaggcaacgt aaacgggcac cactttgtga
1381 tcgacggaga tggtacaggc aagccttatg agggaaaaca gaccatggat cttgaagtca
1441 aagagggcgg acctctgcct tttgcctttg atatcctgac cactgcattc cattacggca
1501 acagggtatt cgtgaaatat ccagacaaca tacaagacta ttttaagcag tcgtttccta
1561 aggggtattc gtgggaacga agcatgactt tcgaagacgg gggcatttgc tatgccagaa
1621 gcgacataac aatggaaggg gacactttct ataataaagt tcgattttat ggtaccaact
1681 ttcccgccaa tggtccagtt atgcagaaga agacgctgaa atggatgccg agctggacgc
1741 gttcatcacg tcgtaagtgg aataagacag atcacgcact gagagctata ggtcggctga
1801 gctcaggagg ttctggaggt tctgattaca aggacgacga tgacaaggga ggttctggag
1861 gttcttaccc atacgacgta ccagattacg ctggaggttc tggaggttct gagcagaagc
1921 tgatctcaga ggaggacctg cgtacgtaaa agcttatcga taatcaacct ctggattaca
1981 aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat
2041 acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct
2101 ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac
2161 gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca
2221 cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca
2281 tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg
2341 tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga
2401 ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt
2461 cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga
2521 gtcggatctc cctttgggcc gcctccccgc atcgataccg tcgacctcga cccgggcggc
2581 cgcttcgagc agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
2641 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
2701 taagctgcaa taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg
2761 gggagatgtg ggaggttttt taaagcaagt aaaacctcta caaatgtggt aaaatcgata
2821 aggatcttcc tagagcatgg ctacgtagat aagtagcatg gcgggttaat cattaactac
2881 aaggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgac
2941 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
3001 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
3061 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
3121 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
3181 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
3241 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
3301 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
3361 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
3421 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
3481 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
3541 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
3601 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
3661 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
3721 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
3781 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
3841 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
3901 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
3961 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
4021 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
4081 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
4141 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
4201 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
4261 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
4321 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
4381 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
4441 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
4501 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
4561 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
4621 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
4681 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
4741 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
4801 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg
4861 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
4921 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
4981 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
5041 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
5101 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
5161 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
5221 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
5281 ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag
5341 ggcgatcggt gcgggcctct tcgctattac gccagctgcg cgctcgctcg ctcactgagg
5401 ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc
5461 gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag ttaatgatta
5521 acccgccatg ctacttatct acgtagccat gctctaggaa gatctct
//
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