| 质粒名称: | sfGFP-Cx43K258stop |
|---|---|
| 出品公司： | addgene |
| 目录编号: | 69025 |
| 存储实验室: | David Spray |
|
载体骨架基本信息 |
|
| 载体名称: | EGFP-C1 |
| 空载体大小: | 5514bp |
| 完整质粒大小: | 5513 bp |
| 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: | Cx43 |
| 物种来源: | 褐家鼠(大鼠) |
| 插入大小: | 768 bp |
| 基因突变: | non-monomerized sfGFP fused in frame to N-terminus of truncated Cx43delete codons 258-381 of the Cx43 CDS |
| 启动子: | CMV |
| 标签/融合蛋白: | super folder GFP (N terminal on insert) |
| 克隆方法: | Ligation Independent Cloning结扎独立克隆 |
| 5’测序引物 | CMV |
| 3’测序引物 | C1N1-rev: ACCTCTACAAATGTGGTATGGCTGATTATG |
|
文献引用方法 |
|
| 材料和方法部分: |
sfGFP-Cx43K258stop was a gift from David Spray (Addgene plasmid # 69025 ; http://n2t.net/addgene:69025 ; RRID:Addgene_69025)
|
| 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 sfGFP-Cx43K258stop 5513 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses rat Cx43 (Gja1 CDS) truncated at amino acid 258 and tagged
with sfGFP (V206-version) on the N-terminus. CMV promoter..
ACCESSION .
VERSION .
KEYWORDS sfGFP-Cx43K258stop
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 5513)
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 5513)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
http://www.biofeng.com
FEATURES Location/Qualifiers
source 1..5513
/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 607..616
/regulatory_class="other"
/note="vertebrate consensus sequence for strong initiation
of translation (Kozak, 1987)"
CDS 613..1329
/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(658..679)
/label=EGFP-N
/note="EGFP, reverse primer"
primer_bind 1266..1287
/label=EGFP-C
/note="EGFP, forward primer"
polyA_signal 2301..2422
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
primer_bind complement(2338..2357)
/label=SV40pA-R
/note="SV40 polyA, reverse primer"
primer_bind 2392..2411
/label=EBV-rev
/note="SV40 polyA terminator, reverse primer"
rep_origin complement(2429..2884)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(2566..2587)
/label=F1ori-F
/note="F1 origin, forward primer"
primer_bind 2778..2797
/label=F1ori-R
/note="F1 origin, reverse primer"
promoter 2911..3015
/gene="bla"
/label=AmpR promoter
promoter 3017..3374
/label=SV40 promoter
/note="SV40 enhancer and early promoter"
primer_bind complement(3040..3060)
/label=pBABE 3'
/note="SV40 enhancer, reverse primer for pBABE vectors"
rep_origin 3225..3360
/label=SV40 ori
/note="SV40 origin of replication"
primer_bind 3287..3306
/label=SV40pro-F
/note="SV40 promoter/origin, forward primer"
CDS 3409..4203
/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(3463..3482)
/label=Neo-R
/note="Neomycin resistance gene, reverse primer"
primer_bind 4073..4092
/label=Neo-F
/note="Neomycin resistance gene, forward primer"
primer_bind complement(4391..4410)
/label=TK-pA-R
/note="Thymidine kinase polyA, reverse primer"
polyA_signal 4435..4482
/label=HSV TK poly(A) signal
/note="herpes simplex virus thymidine kinase
polyadenylation signal (Cole and Stacy, 1985)"
rep_origin 4811..5399
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 5300..5319
/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 gctagcgcta
601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgcgcggcga gggcgagggc
721 gatgccacca acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
841 gaccacatga agcgccacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
901 cgcaccatca gcttcaagga cgacggcacc tacaagaccc gcgccgaggt gaagttcgag
961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
1021 atcctggggc acaagctgga gtacaacttc aacagccaca acgtctatat caccgccgac
1081 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacgtgga ggacggcagc
1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
1201 cccgacaacc actacctgag cacccagtcc gtgctgagca aagaccccaa cgagaagcgc
1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactcacgg catggacgag
1321 ctgtacaagt ccggactcag atctcgagct cagatgggtg actggagtgc cttggggaag
1381 cttctggaca aggtccaagc ctactccacc gctggaggga aggtgtggct gtcagtgctc
1441 ttcatattca gaatcctgct cctggggaca gctgttgagt cagcttgggg tgatgaacag
1501 tctgcctttc gctgtaacac tcaacaacct ggctgcgaaa acgtctgcta tgacaagtcc
1561 ttccccatct ctcacgtgcg cttctgggtc cttcagatca tattcgtgtc tgtgcccacc
1621 ctcctgtact tggcccatgt gttctatgtg atgaggaagg aagagaagct aaacaagaaa
1681 gaagaggagc tcaaagtggc ccagactgac ggggtcaacg tggagatgca cctgaagcag
1741 attgaaatca agaagttcaa gtacgggatt gaagagcacg gcaaggtgaa aatgaggggc
1801 ggcttgctga gaacctacat catcagcatc ctcttcaagt ctgtcttcga ggtggccttc
1861 ctgctcatcc agtggtacat ctatgggttc agcttgagcg cggtctacac ctgcaagaga
1921 gatccctgcc cccaccaggt agactgcttc ctctcacgtc ccacggagaa aaccatcttc
1981 atcatcttca tgctggtggt gtccttggtg tctctcgctt tgaacatcat tgagctcttc
2041 tacgtcttct tcaaaggcgt taaggatcgc gtgaagggaa gaagcgatcc ttaccacgcc
2101 accactggcc cactgagccc atcataagag ctcaagcttc gaattctgca gtcgacggta
2161 ccgcgggccc gggatccacc ggatctagat aactgatcat aatcagccat accacatttg
2221 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
2281 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
2341 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
2401 ccaaactcat caatgtatct taacgcgtaa attgtaagcg ttaatatttt gttaaaattc
2461 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
2521 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
2581 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
2641 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
2701 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
2761 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
2821 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
2881 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
2941 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
3001 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
3061 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
3121 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
3181 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
3241 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
3301 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
3361 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
3421 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
3481 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
3541 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
3601 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
3661 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
3721 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
3781 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
3841 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
3901 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
3961 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
4021 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
4081 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
4141 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc
4201 tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag
4261 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
4321 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg
4381 ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa
4441 taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt
4501 cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg
4561 tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca
4621 acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt tagattgatt
4681 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
4741 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
4801 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
4861 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
4921 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
4981 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
5041 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
5101 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
5161 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
5221 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
5281 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
5341 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
5401 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
5461 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccatg cat
//
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