| 质粒名称: | AAV-CaMKIIa-GCaMP6f-P2A-nls-dTomato |
|---|---|
| 出品公司: | 爱迪基因 |
| 目录编号: | 51087 |
| 存储实验室: | Jonathan Ting |
|
载体骨架基本信息 |
|
| 载体名称: | pAAV-CaMKIIa-WPRE-BGHpA |
| 载体出品公司: | custom |
| 载体抗性: | 氨苄青霉素 |
| 空载体大小: | 5095 bp |
| 完整质粒大小: | 7255 bp |
| 载体修饰: | 改变克隆位点并用较小的BGHpA取代HGHpA |
| 载体类型: | 哺乳细胞表达,AAV |
| 筛选标记: | -- |
| 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 |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子 | CaMKIIa 1.3 kb |
| 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 | ttctccgtttgcactcaggagc |
| 3' 测序引物 | CCATACGGGAAGCAATAGCATG |
|
文献引用方法 |
|
| 材料和方法部分: | AAV-CaMKIIa-GCaMP6f-P2A-nls-dTomato was a gift from Jonathan Ting (Addgene plasmid # 51087 ; http://n2t.net/addgene:51087 ; RRID:Addgene_51087) |
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-CaMKIIa-GCaMP6f-P2A-nls-dTomato 7255 bp ds-DNA circular SYN 06-AUG-2018
DEFINITION Forebrain principle neuron specific expression of GCaMP6f Ca sensor
and physically separate nuclear localized dTomato fluorophore .
ACCESSION .
VERSION .
KEYWORDS AAV-CaMKIIa-GCaMP6f-P2A-nls-dTomato
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 7255)
TITLE Ting GCaMP6 plasmids
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 7255)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Aug 6, 2018 from SnapGene Server 1.1.58
http://www.snapgene.com
FEATURES Location/Qualifiers
source 1..7255
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..141
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 12..141
/label=AAV2 ITR
regulatory 1465..1474
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1471..2820
/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 1567..1576
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
primer_bind 1857..1878
/label=EGFP-C
/note="EGFP, forward primer"
primer_bind complement(1984..2005)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(2245..2264)
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
CDS 2836..2892
/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 2905..2925
/codon_start=1
/product="nuclear localization signal of SV40 (simian virus
40) large T antigen"
/label=SV40 NLS
/translation="PKKKRKV"
CDS 2926..3624
/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(3056..3074)
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind 3324..3343
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 3518..3541
/label=DsRed1-C
/note="DsRed1, forward primer"
misc_feature 3674..4262
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(3727..3747)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(4145..4156)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 4295..4406
/label=bGH poly(A) signal
/note="bovine growth hormone polyadenylation signal"
repeat_region 4518..4658
/label=AAV2 ITR
/note="inverted terminal repeat of adeno-associated virus
serotype 2"
repeat_region 4518..4647
/label=AAV2 ITR
rep_origin 4733..5188
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(4820..4839)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 5030..5051
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind complement(5205..5224)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 5324..5346
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind complement(5384..5402)
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
promoter 5470..5574
/gene="bla"
/label=AmpR promoter
CDS 5575..6435
/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(5793..5812)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 6606..7194
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 7095..7114
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
121 actccatcac taggggttcc tgcggccgca cgcgtttaat taacattatg gccttaggtc
181 acttcatctc catggggttc ttcttctgat tttctagaaa atgagatggg ggtgcagaga
241 gcttcctcag tgacctgccc agggtcacat cagaaatgtc agagctagaa cttgaactca
301 gattactaat cttaaattcc atgccttggg ggcatgcaag tacgatatac agaaggagtg
361 aactcattag ggcagatgac caatgagttt aggaaagaag agtccagggc agggtacatc
421 tacaccaccc gcccagccct gggtgagtcc agccacgttc acctcattat agttgcctct
481 ctccagtcct accttgacgg gaagcacaag cagaaactgg gacaggagcc ccaggagacc
541 aaatcttcat ggtccctctg ggaggatggg tggggagagc tgtggcagag gcctcaggag
601 gggccctgct gctcagtggt gacagatagg ggtgagaaag cagacagagt cattccgtca
661 gcattctggg tctgtttggt acttcttctc acgctaaggt ggcggtgtga tatgcacaat
721 ggctaaaaag cagggagagc tggaaagaaa caaggacaga gacagaggcc aagtcaacca
781 gaccaattcc cagaggaagc aaagaaacca ttacagagac tacaaggggg aagggaagga
841 gagatgaatt agcttcccct gtaaacctta gaacccagct gttgccaggg caacggggca
901 atacctgtct cttcagagga gatgaagttg ccagggtaac tacatcctgt ctttctcaag
961 gaccatccca gaatgtggca cccactagcc gttaccatag caactgcctc tttgccccac
1021 ttaatcccat cccgtctgtt aaaagggccc tatagttgga ggtgggggag gtaggaagag
1081 cgatgatcac ttgtggacta agtttgttcg catccccttc tccaaccccc tcagtacatc
1141 accctggggg aacagggtcc acttgctcct gggcccacac agtcctgcag tattgtgtat
1201 ataaggccag ggcaaagagg agcaggtttt aaagtgaaag gcaggcaggt gttggggagg
1261 cagttaccgg ggcaacggga acagggcgtt tcggaggtgg ttgccatggg gacctggatg
1321 ctgacgaagg ctcgcgaggc tgtgagcagc cacagtgccc tgctcagaag ccccaagctc
1381 gtcagtcaag ccggttctcc gtttgcactc aggagcacgg gcaggcgagt ggcccctagt
1441 tctgggggca gcgggggatc cggcgccacc atgggttctc atcatcatca tcatcatggt
1501 atggctagca tgactggtgg acagcaaatg ggtcgggatc tgtacgacga tgacgataag
1561 gatctcgcca ccatggtcga ctcatcacgt cgtaagtgga ataagacagg tcacgcagtc
1621 agagctatag gtcggctgag ctcactcgag aacgtctata tcaaggccga caagcagaag
1681 aacggcatca aggcgaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
1741 gcctaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
1801 cactacctga gcgtgcagtc caaactttcg aaagacccca acgagaagcg cgatcacatg
1861 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
1921 ggcggtaccg gagggagcat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
1981 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggt
2041 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
2101 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
2161 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacatc
2221 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
2281 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
2341 ggcaacatcc tggggcacaa gctggagtac aacctgccgg accaactgac tgaagagcag
2401 atcgcagaat ttaaagagga attctcccta tttgacaagg acggggatgg gacaataaca
2461 accaaggagc tggggacggt gatgcggtct ctggggcaga accccacaga agcagagctg
2521 caggacatga tcaatgaagt agatgccgac ggtgacggca caatcgactt ccctgagttc
2581 ctgacaatga tggcaagaaa aatgaaatac agggacacgg aagaagaaat tagagaagcg
2641 ttcggtgtgt ttgataagga tggcaatggc tacatcagtg cagcagagct tcgccacgtg
2701 atgacaaacc ttggagagaa gttaacagat gaagaggttg atgaaatgat cagggaagca
2761 gacatcgatg gggatggtca ggtaaactac gaagagtttg tacaaatgat gacagcgaag
2821 gataacggaa gcggagccac taacttctcc ctgttgaaac aagcagggga tgtcgaagag
2881 aatcccgggc catctagaat ggcccccaag aagaagagga aggtggtgag caagggcgag
2941 gaggtcatca aagagttcat gcgcttcaag gtgcgcatgg agggctccat gaacggccac
3001 gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca gaccgccaag
3061 ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc
3121 atgtacggct ccaaggcgta cgtgaagcac cccgccgaca tccccgatta caagaagctg
3181 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggtctggtg
3241 accgtgaccc aggactcctc cctgcaggac ggcacgctga tctacaaggt gaagatgcgc
3301 ggcaccaact tcccccccga cggccccgta atgcagaaga agaccatggg ctgggaggcc
3361 tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg gcgagatcca ccaggccctg
3421 aagctgaagg acggcggcca ctacctggtg gagttcaaga ccatctacat ggccaagaag
3481 cccgtgcaac tgcccggcta ctactacgtg gacaccaagc tggacatcac ctcccacaac
3541 gaggactaca ccatcgtgga acagtacgag cgctccgagg gccgccacca cctgttcctg
3601 tacggcatgg acgagctgta caagtaagtc gacggcgcgc ccctgcaggg aattcgatat
3661 caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
3721 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
3781 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
3841 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
3901 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
3961 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
4021 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
4081 tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
4141 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
4201 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
4261 gcatcgatac cgagcgctgc tcgagagatc tcgactgtgc cttctagttg ccagccatct
4321 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
4381 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
4441 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgcacgt
4501 gcggaccgag cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
4561 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
4621 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
4681 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
4741 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
4801 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
4861 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
4921 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
4981 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
5041 ttccaaactg gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt
5101 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
5161 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
5221 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
5281 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
5341 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
5401 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
5461 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
5521 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
5581 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
5641 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
5701 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
5761 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
5821 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
5881 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
5941 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
6001 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
6061 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
6121 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
6181 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
6241 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
6301 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
6361 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
6421 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
6481 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
6541 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
6601 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
6661 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
6721 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
6781 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
6841 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
6901 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
6961 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
7021 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
7081 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
7141 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
7201 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
