| 质粒名称: | AAV-hSyn1-GCaMP6f-P2A-nls-dTomato |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 51085 |
| 存储实验室: | Jonathan Ting |
|
载体骨架基本信息 |
|
| 载体名称: | pAAV-hSyn1-WPRE-HGHpA |
| 载体出品公司: | Modified from Karl Deisseroth |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 4590 bp |
| 完整质粒大小: | 6750 bp |
| 载体修饰: | 改变克隆位点 |
| 载体类型: | 哺乳动物表达,AAV |
| 高拷贝/低拷贝: | 高拷贝 |
| 生长条件: | 37 ℃ |
|
插入基因信息 |
|
| 插入基因名称: | GCaMP6f,G6f,GCaMP6 fast |
| 物种来源: | Synthetic |
| 插入大小: | 2160 bp |
| 启动子 | human Synapsin1 |
| 标签/融合蛋白: | P2A nls dTomato（插入时的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: | AscI(未破坏) |
| 5' 测序引物 | ctcagcgctgcctcagtctg |
| 3' 测序引物 | CCATACGGGAAGCAATAGCATG |
|
文献引用方法 |
|
| 材料和方法部分: | AAV-hSyn1-GCaMP6f-P2A-nls-dTomato was a gift from Jonathan Ting (Addgene plasmid # 51085 ; http://n2t.net/addgene:51085 ; RRID:Addgene_51085) |
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 AAV-hSyn1-GCaMP6f-P2A-nls-dTomato 6739 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Neuronal expression of GCaMP6f Ca sensor and physically separate
nuclear localized dTomato fluorophore .
ACCESSION .
VERSION .
KEYWORDS AAV-hSyn1-GCaMP6f-P2A-nls-dTomato
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6739)
TITLE Ting GCaMP6 plasmids
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 6739)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..6739
/organism="synthetic DNA construct"
/mol_type="other DNA"
promoter 21..468
/label=hSyn promoter
/note="human synapsin I promoter; confers neuron-specific
expression (Kugler et al., 2003)"
regulatory 495..504
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 501..1850
/codon_start=1
/product="improved fluorescent protein-based calcium sensor
(Chen et al., 2013)"
/label=GCaMP6f
/translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
regulatory 597..606
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
primer_bind 887..908
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(1014..1035)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(1275..1294)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
CDS 1866..1922
/codon_start=1
/product="2A peptide from porcine teschovirus-1
polyprotein"
/label=P2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="ATNFSLLKQAGDVEENPGP"
CDS 1935..1955
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 1956..2654
/codon_start=1
/product="dimeric variant of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=dTomato
/note="mammalian codon-optimized"
/translation="VSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTAK
LKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGL
VTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIHQ
ALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRHH
LFLYGMDELYK"
primer_bind complement(2086..2104)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 2354..2373
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 2548..2571
/label=DsRed1-C
/note="DsRed1, forward primer"
misc_feature 2704..3292
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(2757..2777)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(3175..3186)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 3324..3800
/label=hGH poly(A) signal
/note="human growth hormone polyadenylation signal"
primer_bind complement(3523..3542)
/label=hGH-PA-R
/note="Human growth hormone terminator, reverse primer"
repeat_region 3840..3980
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 3840..3969
/label=AAV2 ITR
rep_origin 4055..4510
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4142..4161)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 4352..4373
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(4527..4546)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 4646..4668
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(4706..4724)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 4792..4896
/gene="bla"
/label=AmpR promoter
CDS 4897..5757
/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(5115..5134)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 5928..6516
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 6417..6436
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
repeat_region 6578..6707
/label=AAV2 ITR (alternate)
/note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
1 tgcagagggc cctgcgtatg agtgcaagtg ggttttagga ccaggatgag gcggggtggg
61 ggtgcctacc tgacgaccga ccccgaccca ctggacaagc acccaacccc cattccccaa
121 attgcgcatc ccctatcaga gagggggagg ggaaacagga tgcggcgagg cgcgtgcgca
181 ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc ctggcggcgc gcgccaccgc
241 cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc ccccgcaaac tccccttccc
301 ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg gaccgcacca cgcgaggcgc
361 gagatagggg ggcacgggcg cgaccatctg cgctgcggcg ccggcgactc agcgctgcct
421 cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga gagcgcagtc gagaaaccgg
481 ctagaggatc cggcgccacc atgggttctc atcatcatca tcatcatggt atggctagca
541 tgactggtgg acagcaaatg ggtcgggatc tgtacgacga tgacgataag gatctcgcca
601 ccatggtcga ctcatcacgt cgtaagtgga ataagacagg tcacgcagtc agagctatag
661 gtcggctgag ctcactcgag aacgtctata tcaaggccga caagcagaag aacggcatca
721 aggcgaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc gcctaccact
781 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
841 gcgtgcagtc caaactttcg aaagacccca acgagaagcg cgatcacatg gtcctgctgg
901 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggcggtaccg
961 gagggagcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
1021 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg
1081 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
1141 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
1201 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc caggagcgca
1261 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
1321 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
1381 tggggcacaa gctggagtac aacctgccgg accaactgac tgaagagcag atcgcagaat
1441 ttaaagagga attctcccta tttgacaagg acggggatgg gacaataaca accaaggagc
1501 tggggacggt gatgcggtct ctggggcaga accccacaga agcagagctg caggacatga
1561 tcaatgaagt agatgccgac ggtgacggca caatcgactt ccctgagttc ctgacaatga
1621 tggcaagaaa aatgaaatac agggacacgg aagaagaaat tagagaagcg ttcggtgtgt
1681 ttgataagga tggcaatggc tacatcagtg cagcagagct tcgccacgtg atgacaaacc
1741 ttggagagaa gttaacagat gaagaggttg atgaaatgat cagggaagca gacatcgatg
1801 gggatggtca ggtaaactac gaagagtttg tacaaatgat gacagcgaag gataacggaa
1861 gcggagccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag aatcccgggc
1921 catctagaat ggcccccaag aagaagagga aggtggtgag caagggcgag gaggtcatca
1981 aagagttcat gcgcttcaag gtgcgcatgg agggctccat gaacggccac gagttcgaga
2041 tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga
2101 ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc atgtacggct
2161 ccaaggcgta cgtgaagcac cccgccgaca tccccgatta caagaagctg tccttccccg
2221 agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggtctggtg accgtgaccc
2281 aggactcctc cctgcaggac ggcacgctga tctacaaggt gaagatgcgc ggcaccaact
2341 tcccccccga cggccccgta atgcagaaga agaccatggg ctgggaggcc tccaccgagc
2401 gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca ccaggccctg aagctgaagg
2461 acggcggcca ctacctggtg gagttcaaga ccatctacat ggccaagaag cccgtgcaac
2521 tgcccggcta ctactacgtg gacaccaagc tggacatcac ctcccacaac gaggactaca
2581 ccatcgtgga acagtacgag cgctccgagg gccgccacca cctgttcctg tacggcatgg
2641 acgagctgta caagtaagtc gacggcgcgc ccctgcaggg aattcgatat caagcttatc
2701 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
2761 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
2821 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
2881 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
2941 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
3001 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
3061 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
3121 ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
3181 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
3241 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac
3301 cgagcgctgc tcgagagatc tacgggtggc atccctgtga cccctcccca gtgcctctcc
3361 tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca
3421 tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga
3481 gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct
3541 ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc
3601 tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat
3661 ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct
3721 aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca
3781 ctgctccctt ccctgtcctt ctgattttgt aggtaaccac gtgcggaccg agcggccgca
3841 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
3901 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
3961 agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg
4021 tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc
4081 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc
4141 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
4201 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
4261 aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
4321 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
4381 ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat
4441 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
4501 tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
4561 ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc
4621 gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca
4681 tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc
4741 atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
4801 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
4861 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
4921 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
4981 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
5041 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
5101 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
5161 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
5221 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
5281 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
5341 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
5401 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
5461 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
5521 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
5581 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
5641 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
5701 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
5761 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
5821 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
5881 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
5941 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
6001 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
6061 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
6121 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
6181 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
6241 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
6301 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
6361 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
6421 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
6481 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
6541 cggttcctgg ccttttgctg gccttttgct cacatgtcct gcaggcagct gcgcgctcgc
6601 tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga
6661 gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg gccgcacgcg
6721 tttaattaag tgtctagac
//
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