| 质粒名称: | pUC57-rm_gCarvi-T2A |
|---|---|
| 出品公司: | Addgene |
| 目录编号: | 190334 |
| 存储实验室: | Naoto Saitoh |
|
载体骨架基本信息 |
|
| 载体名称: | pUC57 |
| 载体出品公司: | Clontech |
| 空载体大小: | 2714 bp |
| 完整质粒大小: | 4748 bp |
| 载体修饰: | -- |
| 载体类型: | -- |
| 筛选标记: | -- |
| 载体抗性: | 氨苄青霉素，100μg/mL |
| 生长温度: | 37 ℃ |
| 宿主菌: | DH5a |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | ratiometric gCarvi |
| 物种来源: | Synthetic |
| 插入大小: | 1947 bp |
| 基因突变: | -- |
| GenBank编号 | -- |
| 启动子: | -- |
| 标签/融合蛋白: | -- |
| 克隆方法: | 限制性内切酶 |
| 5’克隆位点: | BglII (未破坏) |
| 3’克隆位点: | SalI (未破坏) |
| 5’测序引物 | M13 rev |
| 3’测序引物 | M13 fwd |
|
文献引用方法 |
|
| 材料和方法部分: | pUC57-rm_gCarvi-T2A was a gift from Naoto Saitoh (Addgene plasmid # 190334 ; http://n2t.net/addgene:190334 ; RRID:Addgene_190334) |
| 参考文献部分: | Green fluorescent cAMP indicator of high speed and specificity suitable for neuronal live-cell imaging. Kawata S, Mukai Y, Nishimura Y, Takahashi T, Saitoh N. Proc Natl Acad Sci U S A. 2022 Jul 12;119(28):e2122618119. doi: 10.1073/pnas.2122618119. Epub 2022 Jul 6. 10.1073/pnas.2122618119 PubMed 35867738 |
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 pUC57-rm_gCarvi-T2A 4748 bp ds-DNA circular SYN 13-SEP-2022
DEFINITION Ratiometric reporter for cAMP.
ACCESSION .
VERSION .
KEYWORDS pUC57-rm_gCarvi-T2A
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4748)
AUTHORS Kawata S, Mukai Y, Nishimura Y, Takahashi T, Saitoh N
TITLE Green fluorescent cAMP indicator of high speed and specificity
suitable for neuronal live-cell imaging.
JOURNAL Proc Natl Acad Sci U S A. 2022 Jul 12;119(28):e2122618119. doi:
10.1073/pnas.2122618119. Epub 2022 Jul 6.
PUBMED 35867738
REFERENCE 2 (bases 1 to 4748)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported Sep 13, 2022 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..4748
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind complement(29..51)
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
primer_bind 151..170
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 364..386
/label=M13/pUC Forward
/note="In lacZ gene"
primer_bind 378..395
/label=M13 Forward
/note="In lacZ gene. Also called M13-F20 or M13 (-21)
Forward"
primer_bind 379..395
/label=M13 fwd
/note="common sequencing primer, one of multiple similar
variants"
CDS complement(471..524)
/codon_start=1
/product="2A peptide from Thosea asigna virus capsid
protein"
/label=T2A
/note="Eukaryotic ribosomes fail to insert a peptide bond
between the Gly and Pro residues, yielding separate
polypeptides."
/translation="EGRGSLLTCGDVEENPGP"
primer_bind 658..677
/label=EXFP-R
/note="For distinguishing EGFP variants, reverse primer"
primer_bind complement(1044..1065)
/label=EGFP-C
/note="EGFP, forward primer"
CDS complement(1692..1757)
/codon_start=1
/product="three tandem FLAG(R) epitope tags, followed by an
enterokinase cleavage site"
/label=3xFLAG
/translation="DYKDHDGDYKDHDIDYKDDDDK"
regulatory 1757..1766
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS complement(1788..2495)
/codon_start=1
/product="monomeric derivative of DsRed fluorescent protein
(Shaner et al., 2004)"
/label=mCherry
/note="mammalian codon-optimized"
/translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
EGRHSTGGMDELYK"
primer_bind complement(2063..2082)
/label=mCherry-F
/note="mCherry, forward primer"
primer_bind 2332..2350
/label=mCherry-R
/note="mCherry, reverse primer"
primer_bind complement(2527..2543)
/label=M13 rev
/note="common sequencing primer, one of multiple similar
variants"
primer_bind complement(2527..2543)
/label=M13 Reverse
/note="In lacZ gene. Also called M13-rev"
primer_bind complement(2540..2562)
/label=M13/pUC Reverse
/note="In lacZ gene"
protein_bind 2551..2567
/label=lac operator
/bound_moiety="lac repressor encoded by lacI"
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
promoter complement(2575..2605)
/label=lac promoter
/note="promoter for the E. coli lac operon"
protein_bind 2620..2641
/label=CAP binding site
/bound_moiety="E. coli catabolite activator protein"
/note="CAP binding activates transcription in the presence
of cAMP."
primer_bind complement(2758..2775)
/label=L4440
/note="L4440 vector, forward primer"
rep_origin complement(2929..3517)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind complement(3009..3028)
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
CDS complement(3688..4548)
/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 4311..4330
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
promoter complement(4549..4653)
/gene="bla"
/label=AmpR promoter
primer_bind 4721..4739
/label=pBRforEco
/note="pBR322 vectors, upsteam of EcoRI site, forward
primer"
ORIGIN
1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
421 tgcatctaga tcccaatgtc gacggtatcg ataagcttga tatcgaattc agggccaggg
481 ttttcctcca cgtcgccgca ggtcagcagg ctgcctctgc cctcagatcc ctcgacaagc
541 ttactagtgt tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg
601 cccttcagct cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc
661 ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct ggatgtagcc ttcgggcatg
721 gcggacttga agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg
781 ccgtaggtca gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag
841 atgaacttca gggtcagctt gccgtaggtg gcatcgccct caccctcgcc ggacacgctg
901 aacttgtggc cgtttacgtc gccgtccagc tcgacctgga tgggcaccac cccggtgaac
961 agctcctcgc ccttgctcac catgctccct ccggtaccgc ccttgtacag ctcgtccatg
1021 ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc gcgcttctcg
1081 ttggggtctt tcgaaagaat ggactgcacg ctcaggtagt ggttgtcggg cagcagcacg
1141 gggccgtcgc cgatgggggt gttctgctgg tagtggtagg cgagctgcac gccgccgtcc
1201 tcgatgttgt ggcggatctt gaagttcgcc ttgatgccgt tcttctgctt gtcggccttg
1261 atatagacgt tctcgaggta caggttcccc accttctcag aggtaacctg aagccttctc
1321 gccatctggg ctgagaggcg catgagtatg tcgggattca cctgtatgag ttgccggaat
1381 ttcttgtatg agatttcggc cacctcacag gctgtcttag ccctcaccca agcggatctc
1441 tcctgtcctt cctcgaacag gcccagttct ccgataaagt caccctggtt cagatagctc
1501 aggatcattt cctttccttc ttcatcctta atcagcacgg cgactgaacc cttcactata
1561 taatacaggg tttcagcctt ctcgccctga tggatcaggg tgctcttaga tgggtacttg
1621 tgaatgtgac agtgtgacag aaaccactcc aatgttgggt cggtctgtgg tttgcccagc
1681 accatggatc ccttgtcatc gtcatccttg taatcgatgt catgatcttt ataatcaccg
1741 tcatggtctt tgtagtccat ggtggcacta gtagatccga gtccggactt gtacagctcg
1801 tccatgccgc cggtggagtg gcggccctcg gcgcgttcgt actgttccac gatggtgtag
1861 tcctcgttgt gggaggtgat gtccaacttg atgttgacgt tgtaggcgcc gggcagctgc
1921 acgggcttct tggccttgta ggtggtcttg acctcagcgt cgtagtggcc gccgtccttc
1981 agcttcagcc tctgcttgat ctcgcccttc agggcgccgt cctcggggta catccgctcg
2041 gaggaggcct cccagcccat ggtcttcttc tgcattacgg ggccgtcgga ggggaagttg
2101 gtgccgcgca gcttcacctt gtagatgaac tcgccgtcct gcagggagga gtcctgggtc
2161 acggtcacca cgccgccgtc ctcgaagttc atcacgcgct cccacttgaa gccctcgggg
2221 aaggacagct tcaagtagtc ggggatgtcg gcggggtgct tcacgtaggc cttggagccg
2281 tacatgaact gaggggacag gatgtcccag gcgaagggca gggggccacc cttggtcacc
2341 ttcagcttgg cggtctgggt gccctcgtag gggcggccct cgccctcgcc ctcgatctcg
2401 aactcgtggc cgttcacgga gccctccatg tgcaccttga agcgcatgaa ctccttgatg
2461 atggccatgt tatcctcctc gcccttgctc accatggtag atccactagt tctagtagat
2521 ctcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
2581 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
2641 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
2701 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
2761 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
2821 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
2881 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
2941 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
3001 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
3061 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
3121 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
3181 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
3241 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
3301 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
3361 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
3421 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
3481 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
3541 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
3601 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
3661 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
3721 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
3781 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
3841 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
3901 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
3961 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
4021 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
4081 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
4141 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
4201 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
4261 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
4321 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
4381 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
4441 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
4501 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
4561 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
4621 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
4681 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
4741 ctttcgtc
//
