| 质粒名称: | pAAV_hsyn_NES-his-CaMPARI2-F391W-WPRE-SV40 |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 101062 |
| 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: | 5716 bp |
| 载体类型: | AAV |
| 筛选标记: | 新霉素（G418） |
| 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 |
| 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 | f.hsyn |
| 3’测序引物 | r.WPRE |
|
文献引用方法 |
|
| 材料和方法部分: |
pAAV_hsyn_NES-his-CaMPARI2-F391W-WPRE-SV40 was a gift from Eric Schreiter (Addgene plasmid # 101061 ; http://n2t.net/addgene:101061 ; RRID:Addgene_101061)
For viral preps, please replace (Addgene plasmid # 101061) in the above sentence with: (Addgene viral prep # 101061-AAV1)
|
| 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 |
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_hsyn_NES-his-CaMPARI2-F391W-WPRE-SV40 5563 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION AAV vector expressing CaMPARI2_F391W (Kd = 110nM), a
photoconvertible fluorescent protein-based calcium integrator, in
neurons.
ACCESSION .
VERSION .
KEYWORDS pAAV_hsyn_NES-his-CaMPARI2-F391W-WPRE-SV40
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5563)
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 5563)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5563
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 31..478
/label=hSyn promoter
/note="human synapsin I promoter; confers neuron-specific
expression (Kugler et al., 2003)"
CDS 575..592
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 599..1039
/codon_start=1
/product="calcium-binding protein"
/label=calmodulin
/translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAEL
RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVVMMTAK"
CDS 1760..1816
/codon_start=1
/product="calmodulin-binding peptide from avian smooth
muscle myosin light chain kinase"
/label=calmodulin-binding peptide
/translation="RRKWNKTGHALRAIGRLSS"
CDS 1835..1858
/codon_start=1
/product="FLAG(R) epitope tag, followed by an enterokinase
cleavage site"
/label=FLAG
/translation="DYKDDDDK"
CDS 1877..1903
/codon_start=1
/product="HA (human influenza hemagglutinin) epitope tag"
/label=HA
/translation="YPYDVPDYA"
CDS 1922..1951
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
misc_feature 1973..2561
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(2026..2046)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(2444..2455)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
primer_bind complement(2564..2580)
/label=KS primer
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2565..2581)
/label=pBluescriptKS
/note="For pBluescript vector"
polyA_signal 2610..2731
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2621..2640)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 2675..2694
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind complement(2935..2952)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(3106..3694)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(3186..3205)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(3865..4725)
/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 4488..4507
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(4726..4830)
/gene="bla"
/label=AmpR promoter
rep_origin complement(4856..5311)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4993..5014)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 5205..5224
/label=F1ori-R
/note="F1 origin, reverse primer"
repeat_region 5383..5512
/label=AAV2 ITR
ORIGIN
1 ggaagatctc tgcagagggc cctgcgtatg agtgcaagtg ggttttagga ccaggatgag
61 gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc acccaacccc
121 cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga tgcggcgagg
181 cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc
241 gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac
301 tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca
361 cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc
421 agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga gagcgcagtc
481 gaattcaagc tgctagcaag gatccgccac catgctgcag aacgagcttg ctcttaagtt
541 ggctggactt gatattaaca agactggagg ttctcatcat caccaccacc atggatccga
601 ccaactgact gaagagcaga tcgcagaatt taaagaggct ttctccctat ttgacaagga
661 cggggatggg acaataacaa ccaaggagct ggggacggtg atgcggtctc tggggcagaa
721 ccccacagaa gcagagctgc aggacatgat caatgaagta gatgccgacg gtgacggcac
781 aatcgacttc cctgagttcc tgacaatgat ggcaagaaaa atgaaagaca cagacagtga
841 agaagaaatt agagaagcgt tccgtgtgtt tgataaggat ggcaatggct acatcagtgc
901 agcagagctt cgccacgtga tgacaaacct tggagagaag ttaacagatg aagaggttga
961 tgaaatgatc agggaagcag acatcgatgg ggatggtcag gtaaactacg aagagtttgt
1021 agtgatgatg acagcgaagc tcgagtgcga gaaaatctat gtgcgtgatg gagtgctgac
1081 gggtgatatt catatgatct tgttgcttga aggaaatgcc cattaccgat gtgacttcag
1141 aactacttac aaagctaagg agaagggtgt caagttacca ggcgtgcact atgtggacca
1201 caccattgag attttaagcc atgacaaaga ttacaacaag gttaagatct atgagtatgc
1261 tgttgctcat tctggattgc ctgacaatgc cagacgaggc ggtaccggcg gatccatggt
1321 gagtgcgatt aagccagaca tgaagatcaa actgcgtatg gaaggcaacg taaacgggca
1381 ccactttgtg atcgacggag atggtacagg caagccttat gagggaaaac agaccatgga
1441 tcttgaagtc aaagagggcg gacctctgcc ttttgccttt gatatcctga ccactgcatt
1501 ccattacggc aacagggtat tcgtgaaata tccagacaac atacaagact attttaagca
1561 gtcgtttcct aaggggtatt cgtgggaacg aagcatgact ttcgaagacg ggggcatttg
1621 ctatgccaga agcgacataa caatggaagg ggacactttc tataataaag ttcgatttta
1681 tggtaccaac tttcccgcca atggtccagt tatgcagaag aagacgctga aatggatgcc
1741 gagctggacg cgttcatcac gtcgtaagtg gaataagaca ggtcacgcac tgagagctat
1801 aggtcggctg agctcaggag gttctggagg ttctgattac aaggacgacg atgacaaggg
1861 aggttctgga ggttcttacc catacgacgt accagattac gctggaggtt ctggaggttc
1921 tgagcagaag ctgatctcag aggaggacct gcgtacgtaa aagcttatcg ataatcaacc
1981 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
2041 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
2101 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
2161 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
2221 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
2281 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
2341 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
2401 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
2461 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
2521 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacctcg
2581 acccgggcgg ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac
2641 aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt
2701 tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt
2761 tcaggttcag ggggagatgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg
2821 taaaatcgat aaggatcttc ctagagcatg gctacgtaga taagtagcat ggcgggttaa
2881 tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct
2941 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
3001 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
3061 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
3121 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
3181 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
3241 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
3301 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
3361 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
3421 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
3481 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
3541 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
3601 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
3661 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
3721 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
3781 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
3841 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
3901 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
3961 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
4021 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
4081 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
4141 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
4201 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
4261 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
4321 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
4381 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
4441 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
4501 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
4561 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
4621 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
4681 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
4741 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
4801 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt
4861 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
4921 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
4981 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
5041 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
5101 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
5161 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
5221 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
5281 gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt
5341 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctgcgcgc tcgctcgctc
5401 actgaggccg cccgggcaaa gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg
5461 agcgagcgag cgcgcagaga gggagtggcc aactccatca ctaggggttc cttgtagtta
5521 atgattaacc cgccatgcta cttatctacg tagccatgct cta
//
