| 质粒名称: | pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE |
|---|---|
| 出品公司: | addgene |
| 目录编号: | 104495 |
| 存储实验室: | Douglas Kim, GENIE Project |
|
载体骨架基本信息 |
|
| 载体名称: | AAV-CAG-FLEX |
| 载体出品公司: | Scott Sternson |
| 空载体大小: | 5027 bp |
| 完整质粒大小: | 6379 bp |
| 载体类型: | 哺乳细胞表达 |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 30 ℃ |
| 宿主菌: | NEB Stable |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | jGCaMP7s/GCaMP3-A52V K78H T302L R303P A317L D380Y T381R S383T R392G/GCaMP3 variant 1473/Janelia GCaMP7 |
| 物种来源: | R. norvegicus (rat), G. gallus (chicken); A. victoria (jellyfish) |
| 插入大小: | 1353bp |
| 启动子: | CAG |
| 标签/融合蛋白: | T7 epitope, Xpress tag, 6xHis |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BsmBI(未破坏) |
| 3’克隆位点: | BsmBI(未破坏) |
| 5’测序引物 | GGTTCGGCTTCTGGCGTGTGACC |
|
文献引用方法 |
|
| 材料和方法部分: |
pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE was a gift from Douglas Kim & GENIE Project (Addgene plasmid # 104495 ; http://n2t.net/addgene:104495 ; RRID:Addgene_104495)
For viral preps, please replace (Addgene plasmid # 104495) in the above sentence with: (Addgene viral prep # 104495-AAV1) or (Addgene viral prep # 104495-AAVrg)
|
| 参考文献部分: | High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Dana H, Sun Y, Mohar B, Hulse BK, Kerlin AM, Hasseman JP, Tsegaye G, Tsang A, Wong A, Patel R, Macklin JJ, Chen Y, Konnerth A, Jayaraman V, Looger LL, Schreiter ER, Svoboda K, Kim DS. Nat Methods. 2019 Jul;16(7):649-657. doi: 10.1038/s41592-019-0435-6. Epub 2019 Jun 17. 10.1038/s41592-019-0435-6 PubMed 31209382 |
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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 pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE 6379 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION AAV-mediated expression of ultrasensitive protein calcium sensor
under the CAG promoter, Cre mediated flip-excision switch.
ACCESSION .
VERSION .
KEYWORDS pGP-AAV-CAG-FLEX-jGCaMP7s-WPRE
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 6379)
AUTHORS Dana H, Sun Y, Mohar B, Hulse BK, Kerlin AM, Hasseman JP, Tsegaye G,
Tsang A, Wong A, Patel R, Macklin JJ, Chen Y, Konnerth A, Jayaraman
V, Looger LL, Schreiter ER, Svoboda K, Kim DS
TITLE High-performance calcium sensors for imaging activity in neuronal
populations and microcompartments.
JOURNAL Nat Methods. 2019 Jul;16(7):649-657. doi: 10.1038/s41592-019-0435-6.
Epub 2019 Jun 17.
PUBMED 31209382
REFERENCE 2 (bases 1 to 6379)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..6379
/organism="synthetic DNA construct"
/mol_type="other DNA"
repeat_region 1..130
/label=AAV2 ITR
promoter 441..718
/label=chicken beta-actin promoter
primer_bind 997..1019
/label=pCAGGS-5
/note="Chimeric intron in CAG promoter, forward primer"
primer_bind 1081..1100
/label=pCAG-F
/note="Rabbit beta-globin intron, for pCAG plasmids,
forward primer"
protein_bind complement(1135..1168)
/label=lox2272
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GGATACTT). lox2272 sites are compatible with each
other, but incompatible with loxP or loxN sites (Lee and
Saito, 1988)."
protein_bind complement(1231..1264)
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
CDS complement(1275..2627)
/codon_start=1
/product="improved fluorescent protein-based calcium sensor
(Chen et al., 2013)"
/label=GCaMP6s
/translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
TGHAVRVIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKELFSLFDKDGDGTITTKELGTVMRS
LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
primer_bind 1834..1853
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind 2093..2114
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind complement(2220..2241)
/label=EGFP-C
/note="EGFP, forward primer"
regulatory 2522..2531
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
regulatory 2624..2633
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
protein_bind 2644..2677
/label=lox2272
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GGATACTT). lox2272 sites are compatible with each
other, but incompatible with loxP or loxN sites (Lee and
Saito, 1988)."
protein_bind 2748..2781
/label=loxP
/bound_moiety="Cre recombinase"
/note="Cre-mediated recombination occurs in the 8-bp core
sequence (GCATACAT)."
misc_feature 2823..3411
/label=WPRE
/note="woodchuck hepatitis virus posttranscriptional
regulatory element"
primer_bind complement(2876..2896)
/label=WPRE-R
/note="WPRE, reverse primer"
CDS complement(3294..3305)
/codon_start=1
/product="Factor Xa recognition and cleavage site"
/label=Factor Xa site
/translation="IEGR"
polyA_signal 3454..3575
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(3465..3484)
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
primer_bind 3519..3538
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
repeat_region 3737..3866
/label=AAV2 ITR
primer_bind complement(3932..3949)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(4103..4691)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(4183..4202)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(4862..5722)
/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 5485..5504
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(5723..5827)
/gene="bla"
/label=AmpR promoter
rep_origin complement(5853..6308)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(5990..6011)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 6202..6221
/label=F1ori-R
/note="F1 origin, reverse primer"
ORIGIN
1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
121 aggggttcct tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct
181 aggaagagta ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac
241 tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca
301 agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg
361 gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt
421 agtcatcgct attaccatgg tcgaggtgag ccccacgttc tgcttcactc tccccatctc
481 ccccccctcc ccacccccaa ttttgtattt atttattttt taattatttt gtgcagcgat
541 gggggcgggg gggggggggg ggcgcgcgcc aggcggggsg gggsggggcg aggggcgggg
601 cggsgcgwgg cggagaggtg cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc
661 ttttatggcg aggcggcggc ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg
721 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc
781 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
841 gggctgtaat tagcgcttgg tttaatgacg gcttgtttct tttctgtggc tgcgtgaaag
901 ccttgagggg ctccgggagg gccctttgtg cggggggagc ggctcggggc tgtccgcggg
961 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
1021 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
1081 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattggat ccccataact
1141 tcgtataaag tatcctatac gaagttatat caaaatagga agaccaatgc ttcaccatcg
1201 acccgaattg ccaagcatca ccatcgaccc ataacttcgt ataatgtatg ctatacgaag
1261 ttatactaga gctttcactt cgctgtcatc atttgtacaa actcttcgta gtttacctga
1321 ccatccccat cgatgtctgc ttccctgatc atttcatcaa cctcttcatc tgttaacttc
1381 tctccaaggt ttgtcatcac gtggcgaagc tctgctgcac tgatgtagcc attgccatcc
1441 ttatcaaaca caccgaacgc ttctctaatt tcttcttccg tgtccctgta tttcattttt
1501 cttgccatca ttgtcaggaa ctcagggaag tcgattgtgc cgtcaccgtc ggcatctact
1561 tcattgatca tgtcctgcag ctctgcttct gtggggttct gccccagaga ccgcatcacc
1621 gtccccagct ccttggttgt tattgtccca tccccgtcct tgtcaaatag ggagaaaagc
1681 tctttaaatt ctgcgatctg ctcttcagtc agttggtcag gaaggttgta ctccagcttg
1741 tgccccagga tgttgccgtc ctccttgaag tcgatgccct taagctcgat gcggttcacc
1801 agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag
1861 aagatggtgc gctcctggat gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc
1921 ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg
1981 gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg
2041 taggtggcat cgccctcacc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg
2101 tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg
2161 ctccctccgg taccgccctt gtacagctcg tccatgccga gagtgatccc ggcggcggtc
2221 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttcga aagtttggac
2281 tgcacgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc
2341 tgctggtagt ggtaggcgag ctgcacgccg ccgtcctcga tgttgtggcg gatgtggaag
2401 ttcgccttga tgccgttctt ctgcttgtcg gccttgatat agacgttctc gagtgagctc
2461 agccgaccta tcactctgac tgcgtgacct gtcttattcc acttacgacg tgatgagtcg
2521 accatggtgg cgagatcctt atcgtcatcg tcgtacagat cccgacccat ttgctgtcca
2581 ccagtcatgc tagccatacc atgatgatga tgatgatgag aacccatggt ggcggatcct
2641 agtataactt cgtataggat actttatacg aagttatcat tgggattctt cctattttga
2701 tccaagcatc accatcgacc ctctagtcca gatctcacca tcgacccata acttcgtata
2761 gcatacatta tacgaagtta tgtccctcga agaggttcga attcgatatc aagcttatcg
2821 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
2881 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc
2941 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt
3001 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca
3061 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc
3121 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc
3181 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc
3241 tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc
3301 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc
3361 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc
3421 gtcgacccgg gcggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa
3481 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
3541 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
3601 tgtttcaggt tcagggggag atgtgggagg ttttttaaag caagtaaaac ctctacaaat
3661 gtggtaaaat cgataaggat cttcctagag catggctacg tagataagta gcatggcggg
3721 ttaatcatta actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct
3781 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
3841 gcctcagtga gcgagcgagc gcgcagctgc attaatgaat cggccaacgc gcggggagag
3901 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
3961 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
4021 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
4081 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
4141 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
4201 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
4261 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
4321 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
4381 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
4441 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
4501 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
4561 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
4621 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
4681 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
4741 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
4801 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
4861 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
4921 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
4981 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
5041 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
5101 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
5161 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
5221 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
5281 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
5341 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
5401 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
5461 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
5521 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
5581 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
5641 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
5701 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
5761 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
5821 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
5881 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
5941 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
6001 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
6061 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
6121 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
6181 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
6241 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
6301 gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc
6361 ctcttcgcta ttacgccag
//
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