| 质粒名称: | Cx26-sfGFP |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 69026 |
| 存储实验室: | David Spray |
|
载体骨架基本信息 |
|
| 载体名称: | EGFP-N1 |
| 空载体大小: | 5350 bp |
| 完整质粒大小: | 5350bp |
| 载体类型: | 哺乳细胞表达 |
| 筛选标记: | 新霉素（G418） |
| 载体抗性: | 氨苄霉素 |
| 生长温度: | 37 ℃ |
| 宿主菌: | JM109 |
| 拷贝数: | 高拷贝 |
|
插入基因信息 |
|
| 插入基因名称: | Cx26 |
| 物种来源: | R. norvegicus (rat) |
| 启动子: | CMV |
| 标签/融合蛋白: | non-monomerized superfolder GFP (V206) (C terminal on insert） |
| 克隆方法: | Ligation Independent Cloning |
| 5’测序引物 | CMV-F |
| 3’测序引物 | C1N1-rev: ACCTCTACAAATGTGGTATGGCTGATTATG |
|
文献引用方法 |
|
| 材料和方法部分: |
Cx26-sfGFP was a gift from David Spray (Addgene plasmid # 69026 ; http://n2t.net/addgene:69026 ; RRID:Addgene_69026)
|
| 参考文献部分: | Connexin Type and Fluorescent Protein-fusion Tag Determine Structural Stability of Gap Junction Plaques. Stout RF Jr, Snapp EL, Spray DC. J Biol Chem. 2015 Aug 11. pii: jbc.M115.659979. 10.1074/jbc.M115.659979 PubMed 26265468 |
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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 Cx26-sfGFP 5350 bp ds-DNA circular SYN 13-MAY-2021
DEFINITION Expresses rat Cx26 (Gjb2 CDS) with a 7 amino acid linker on the
C-terminus of the Connexin linking to non-monomerized superfolder
GFP. Expression driven by CMV promoter..
ACCESSION .
VERSION .
KEYWORDS Cx26-sfGFP
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5350)
AUTHORS Stout RF Jr, Snapp EL, Spray DC
TITLE Connexin Type and Fluorescent Protein-fusion Tag Determine
Structural Stability of Gap Junction Plaques.
JOURNAL J Biol Chem. 2015 Aug 11. pii: jbc.M115.659979.
PUBMED 26265468
REFERENCE 2 (bases 1 to 5350)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5350
/organism="synthetic DNA construct"
/mol_type="other DNA"
enhancer 61..364
/label=CMV enhancer
/note="human cytomegalovirus immediate early enhancer"
promoter 365..568
/label=CMV promoter
/note="human cytomegalovirus (CMV) immediate early
promoter"
primer_bind 519..539
/label=CMV-F
/note="Human CMV immediate early promoter, forward primer"
regulatory 1290..1299
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 1296..2015
/codon_start=1
/product="GFP variant that folds robustly even when fused
to poorly folded proteins (Pedelacq et al., 2006)"
/label=superfolder GFP
/note="mammalian codon-optimized"
/translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLL
EFVTAAGITHGMDELYK"
primer_bind complement(1341..1362)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind 1949..1970
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 2138..2259
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2175..2194)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 2229..2248
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(2266..2721)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2403..2424)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 2615..2634
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 2748..2852
/gene="bla"
/label=AmpR promoter
promoter 2854..3211
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(2877..2897)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 3062..3197
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3124..3143
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3246..4040
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
primer_bind complement(3300..3319)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 3910..3929
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(4228..4247)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 4272..4319
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4648..5236
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5137..5156
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
ORIGIN
1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcatgg
601 attggggcac actacagagc atcctcgggg gtgtcaacaa gcactccacc agcattggga
661 aaatctggct cactgtcctc ttcatcttcc gcatcatgat cctcgtggtg gccgcgaagg
721 aggtgtgggg agatgagcaa gccgattttg tttgcaacac tctccagcct ggctgtaaga
781 atgtgtgcta cgaccactac ttccccatct ctcacatccg gctctgggct ctgcagctga
841 tcatggtgtc cacgccggcc ctcctggtag ctatgcacgt ggcctaccgg agacacgaaa
901 agaaacggaa gttcatgaag ggagagataa agaacgagtt taaggacatc gaagagatca
961 aaacccagaa ggtccgtatc gaagggtccc tgtggtggac ctacaccacc agcatcttct
1021 tccgggtcat cttcgaagct gtcttcatgt atgtctttta catcatgtac aatggcttct
1081 tcatgcagcg tctggtgaag tgtaacgcct ggccttgtcc caatacagtg gactgcttca
1141 tttccaggcc cacagaaaag actgtcttca cggtgttcat gatctctgtg tctggaattt
1201 gcatcctgct aaacatcaca gagctgtgct atctgttcat taggtattgc tcagggaagt
1261 ccaaaagacc agtcgcggat ccaccggtcg ccaccatggt gagcaagggc gaggagctgt
1321 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
1381 gcgtgcgcgg cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg aagttcatct
1441 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg
1501 tgcagtgctt cagccgctac cccgaccaca tgaagcgcca cgacttcttc aagtccgcca
1561 tgcccgaagg ctacgtccag gagcgcacca tcagcttcaa ggacgacggc acctacaaga
1621 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
1681 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac ttcaacagcc
1741 acaacgtcta tatcaccgcc gacaagcaga agaacggcat caaggccaac ttcaagatcc
1801 gccacaacgt ggaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
1861 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag tccgtgctga
1921 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
1981 ggatcactca cggcatggac gagctgtaca agtaaagcgg ccgcgactct agatcataat
2041 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
2101 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
2161 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
2221 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
2281 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
2341 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
2401 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
2461 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
2521 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
2581 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
2641 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
2701 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
2761 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
2821 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
2881 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
2941 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
3001 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
3061 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
3121 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
3181 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
3241 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
3301 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
3361 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
3421 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
3481 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
3541 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
3601 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
3661 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
3721 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
3781 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
3841 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
3901 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
3961 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
4021 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
4081 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
4141 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
4201 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
4261 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
4321 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
4381 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
4441 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
4501 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
4561 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
4621 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
4681 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
4741 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
4801 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
4861 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
4921 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
4981 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
5041 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
5101 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
5161 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
5221 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
5281 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
5341 cgccatgcat
//
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