| 出品公司: | Clontech |
|---|---|
| 载体名称: | pAcGFP1-C In-Fusion Ready |
| 质粒类型: | 哺乳动物细胞表达载体；荧光报告载体 |
| 高拷贝/低拷贝: | 高拷贝 |
| 克隆方法: | 限制性内切酶，多克隆位点 |
| 启动子: | CMV IE |
| 载体大小: | 4711 bp |
| 5' 测序引物及序列: | -- |
| 3' 测序引物及序列: | -- |
| 载体标签: | AcGFP1 (N-端） |
| 载体抗性: | 卡那霉素 |
| 筛选标记: | 新霉素（Neomycin） |
| 克隆菌株: | DH5α , HB101 |
| 宿主细胞（系）: | 常规细胞系（293、CV-1、CHO等） |
| 备注: | 哺乳动物载体pAcGFP1-C In-Fusion Ready是线性化的载体，可直接用于片段连接。 |
| 产品目录号: | 634306 |
| 稳定性: | 稳表达 或 瞬表达 |
| 组成型/诱导型: | 组成型 |
| 病毒/非病毒: | 非病毒 |
买家导航
pAcGFP1-C In-Fusion Ready载体质粒基本信息
pAcGFP1-C In-Fusion Ready质粒图谱载体图谱和pAcGFP1-C In-Fusion Ready载体序列质粒序列多克隆位点信息
pAcGFP1-C In-Fusion Ready质粒载体简介
pAcGFP1-C In-Fusion Ready vector is a linearized mammalian expression vector that encodes a Green Fluorescent Protein (GFP) from Aequorea coerulescens. This fluorescent protein coding sequence in this construct has been human codon-optimized for efficient expression and enhanced brightness. AcGFP1 protein has an excitation maximum at 475 nm and an emission maximum at 505 nm. Sequences flanking AcGFP1 have been converted to a Kozak consensus translation initiation site (1) to further increase the translation efficiency in eukaryotic cells. The linearized vector allows direct cloning of PCR products without any need for restriction digestion when using Clontech's
In-Fusion HD Cloning Plus. This is accomplished by the use of a specific 15 nucleotide long sequence within the sense and antisense primers that overlap with the cut ends created by initial digestion of the vector with Sal I and Hind III
The primers that will be used to amplify In-Fusion Ready PCR products require the following 15 nucleotides on their 5’ends:
Sense primer: 5’ AAGGCCTCTGTCGAC followed by sequence of amplification target 3’
Antisense primer: 5’ AGAATTCGCAAGCTT followed by sequence of amplification target 3’
If the gene of interest is added in-frame immediately after the 15 nucleotides mentioned above, this sequence will automatically be in-frame with the AcGFP1 sequence upstream and therefore be expressed as a fusion protein to the C-terminus of AcGFP1.
SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3’ end of the mRNA transcript. In addition, the vector also contains a SV40 origin of replication in mammalian cells expressing the SV40 T-antigen. A neomycin resistance cassette (Neor), containing an SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, andpolyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene, allow selection of stable transformants in eukaryotic cells using G418. A bacterial promoter upstream of the gene expresses kanamycin resistance in E. coli. The pAcGFP1-C In-Fusion Ready Vector also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
Fusions to the C-terminus of AcGFP1 retain the fluorescent properties of the native protein and allow monitoring of the fusion protein localization in vivo. The target gene can be fused into pAcGFP1-C In-Fusion Ready Vector so that it is in-frame with the AcGFP1 coding sequence with no intervening in-frame stop codons. The recombinant AcGFP1 vector can be transfected into mammalian cells using any standard transfection method. Stable transformants can be selected using G418 (2).
Propagation in E. coli
Suitable host strains: Stellar Competent Cells.
Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
E. coli replication origin: pUC
Copy number: ~ 500
Plasmid incompatibility group: pMB1/ColE1
pAcGFP1-C In-Fusion Ready质粒序列载体序列
LOCUS pAcGFP1-C In-Fusion Ready 4711 bp DNA SYN
DEFINITION pAcGFP1-C In-Fusion Ready
ACCESSION
KEYWORDS
SOURCE
ORGANISM other sequences; artificial sequences; vectors.
FEATURES Location/Qualifiers
source 1..4711
/organism="pAcGFP1-C In-Fusion Ready"
/mol_type="other DNA"
terminator 40..268
/label="SV40_PA_terminator"
misc_feature 238..257
/label="EBV_rev_primer"
rep_origin complement(407..713)
/label="f1_origin"
promoter 792..820
/label="AmpR_promoter"
misc_feature complement(886..906)
/label="pBABE_3_primer"
misc_feature complement(892..1107)
/label="SV40_enhancer"
promoter 904..1172
/label="SV40_promoter"
rep_origin 1071..1148
/label="SV40_origin"
misc_feature 1133..1152
/label="SV40pro_F_primer"
CDS 1255..2049
/label="ORF frame 1"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
gene 1258..2046
/label="NeoR/KanR"
/gene="NeoR/KanR"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR
PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL
LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY
QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
CDS complement(1564..2100)
/label="ORF frame 2"
/translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
PPHCISHDGYFLGRSKVR*"
terminator 2224..2493
/label="TK_PA_terminator"
/translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
PPHCISHDGYFLGRSKVR*"
rep_origin 2641..3260
/label="pBR322_origin"
/translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
PPHCISHDGYFLGRSKVR*"
promoter 3369..3921
/label="CMV_immearly_promoter"
/translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
PPHCISHDGYFLGRSKVR*"
misc_feature 3424..3711
/label="CAG_enhancer"
/translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
PPHCISHDGYFLGRSKVR*"
misc_feature 3878..3898
/label="CMV_fwd_primer"
/translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
PPHCISHDGYFLGRSKVR*"
promoter 3879..3948
/label="CMV_promoter"
/translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA
VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA
VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ
PPHCISHDGYFLGRSKVR*"
CDS 3972..34
/label="ORF frame 3"
/translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLT
LKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFE
DDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKN
GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
MIYFGFVTAAAITHGMDELYKSGLKASVEACEFWRIHRI*"
gene 3975..4688
/label="AcGFP1"
/gene="AcGFP1"
/translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLT
LKFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFE
DDGNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKN
GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDH
MIYFGFVTAAAITHGMDELYKSGLKASVEACEFWRIHRI*"
CDS complement(4188..46)
/label="ORF frame 1"
/translation="MISYLDPVDPPEFASFDRGLKSGLVQLIHAVGDGGGGHEAEVDH
VIALLVGVLGQGALGGQVVVIGQQHRAIADGGILLVVVGQLHAAILDVVADLEVHLDA
ILGLVGHDVHIVGVVVVLHLIAQDVAILLEIGAGQLDAIHQGIALELHLGARLVVAVI
LEEDGALLDVALRHGALEEVVLLHVIGVA*"
ORIGIN
1 AGCTTGCGAA TTCTGGCGGA TCCACCGGAT CTAGATAACT GATCATAATC AGCCATACCA
61 CATTTGTAGA GGTTTTACTT GCTTTAAAAA ACCTCCCACA CCTCCCCCTG AACCTGAAAC
121 ATAAAATGAA TGCAATTGTT GTTGTTAACT TGTTTATTGC AGCTTATAAT GGTTACAAAT
181 AAAGCAATAG CATCACAAAT TTCACAAATA AAGCATTTTT TTCACTGCAT TCTAGTTGTG
241 GTTTGTCCAA ACTCATCAAT GTATCTTAAC GCGTAAATTG TAAGCGTTAA TATTTTGTTA
301 AAATTCGCGT TAAATTTTTG TTAAATCAGC TCATTTTTTA ACCAATAGGC CGAAATCGGC
361 AAAATCCCTT ATAAATCAAA AGAATAGACC GAGATAGGGT TGAGTGTTGT TCCAGTTTGG
421 AACAAGAGTC CACTATTAAA GAACGTGGAC TCCAACGTCA AAGGGCGAAA AACCGTCTAT
481 CAGGGCGATG GCCCACTACG TGAACCATCA CCCTAATCAA GTTTTTTGGG GTCGAGGTGC
541 CGTAAAGCAC TAAATCGGAA CCCTAAAGGG AGCCCCCGAT TTAGAGCTTG ACGGGGAAAG
601 CCGGCGAACG TGGCGAGAAA GGAAGGGAAG AAAGCGAAAG GAGCGGGCGC TAGGGCGCTG
661 GCAAGTGTAG CGGTCACGCT GCGCGTAACC ACCACACCCG CCGCGCTTAA TGCGCCGCTA
721 CAGGGCGCGT CAGGTGGCAC TTTTCGGGGA AATGTGCGCG GAACCCCTAT TTGTTTATTT
781 TTCTAAATAC ATTCAAATAT GTATCCGCTC ATGAGACAAT AACCCTGATA AATGCTTCAA
841 TAATATTGAA AAAGGAAGAG TCCTGAGGCG GAAAGAACCA GCTGTGGAAT GTGTGTCAGT
901 TAGGGTGTGG AAAGTCCCCA GGCTCCCCAG CAGGCAGAAG TATGCAAAGC ATGCATCTCA
961 ATTAGTCAGC AACCAGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA
1021 GCATGCATCT CAATTAGTCA GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC
1081 TAACTCCGCC CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG
1141 CAGAGGCCGA GGCCGCCTCG GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTG
1201 GAGGCCTAGG CTTTTGCAAA GATCGATCAA GAGACAGGAT GAGGATCGTT TCGCATGATT
1261 GAACAAGATG GATTGCACGC AGGTTCTCCG GCCGCTTGGG TGGAGAGGCT ATTCGGCTAT
1321 GACTGGGCAC AACAGACAAT CGGCTGCTCT GATGCCGCCG TGTTCCGGCT GTCAGCGCAG
1381 GGGCGCCCGG TTCTTTTTGT CAAGACCGAC CTGTCCGGTG CCCTGAATGA ACTGCAAGAC
1441 GAGGCAGCGC GGCTATCGTG GCTGGCCACG ACGGGCGTTC CTTGCGCAGC TGTGCTCGAC
1501 GTTGTCACTG AAGCGGGAAG GGACTGGCTG CTATTGGGCG AAGTGCCGGG GCAGGATCTC
1561 CTGTCATCTC ACCTTGCTCC TGCCGAGAAA GTATCCATCA TGGCTGATGC AATGCGGCGG
1621 CTGCATACGC TTGATCCGGC TACCTGCCCA TTCGACCACC AAGCGAAACA TCGCATCGAG
1681 CGAGCACGTA CTCGGATGGA AGCCGGTCTT GTCGATCAGG ATGATCTGGA CGAAGAGCAT
1741 CAGGGGCTCG CGCCAGCCGA ACTGTTCGCC AGGCTCAAGG CGAGCATGCC CGACGGCGAG
1801 GATCTCGTCG TGACCCATGG CGATGCCTGC TTGCCGAATA TCATGGTGGA AAATGGCCGC
1861 TTTTCTGGAT TCATCGACTG TGGCCGGCTG GGTGTGGCGG ACCGCTATCA GGACATAGCG
1921 TTGGCTACCC GTGATATTGC TGAAGAGCTT GGCGGCGAAT GGGCTGACCG CTTCCTCGTG
1981 CTTTACGGTA TCGCCGCTCC CGATTCGCAG CGCATCGCCT TCTATCGCCT TCTTGACGAG
2041 TTCTTCTGAG CGGGACTCTG GGGTTCGAAA TGACCGACCA AGCGACGCCC AACCTGCCAT
2101 CACGAGATTT CGATTCCACC GCCGCCTTCT ATGAAAGGTT GGGCTTCGGA ATCGTTTTCC
2161 GGGACGCCGG CTGGATGATC CTCCAGCGCG GGGATCTCAT GCTGGAGTTC TTCGCCCACC
2221 CTAGGGGGAG GCTAACTGAA ACACGGAAGG AGACAATACC GGAAGGAACC CGCGCTATGA
2281 CGGCAATAAA AAGACAGAAT AAAACGCACG GTGTTGGGTC GTTTGTTCAT AAACGCGGGG
2341 TTCGGTCCCA GGGCTGGCAC TCTGTCGATA CCCCACCGAG ACCCCATTGG GGCCAATACG
2401 CCCGCGTTTC TTCCTTTTCC CCACCCCACC CCCCAAGTTC GGGTGAAGGC CCAGGGCTCG
2461 CAGCCAACGT CGGGGCGGCA GGCCCTGCCA TAGCCTCAGG TTACTCATAT ATACTTTAGA
2521 TTGATTTAAA ACTTCATTTT TAATTTAAAA GGATCTAGGT GAAGATCCTT TTTGATAATC
2581 TCATGACCAA AATCCCTTAA CGTGAGTTTT CGTTCCACTG AGCGTCAGAC CCCGTAGAAA
2641 AGATCAAAGG ATCTTCTTGA GATCCTTTTT TTCTGCGCGT AATCTGCTGC TTGCAAACAA
2701 AAAAACCACC GCTACCAGCG GTGGTTTGTT TGCCGGATCA AGAGCTACCA ACTCTTTTTC
2761 CGAAGGTAAC TGGCTTCAGC AGAGCGCAGA TACCAAATAC TGTTCTTCTA GTGTAGCCGT
2821 AGTTAGGCCA CCACTTCAAG AACTCTGTAG CACCGCCTAC ATACCTCGCT CTGCTAATCC
2881 TGTTACCAGT GGCTGCTGCC AGTGGCGATA AGTCGTGTCT TACCGGGTTG GACTCAAGAC
2941 GATAGTTACC GGATAAGGCG CAGCGGTCGG GCTGAACGGG GGGTTCGTGC ACACAGCCCA
3001 GCTTGGAGCG AACGACCTAC ACCGAACTGA GATACCTACA GCGTGAGCTA TGAGAAAGCG
3061 CCACGCTTCC CGAAGGGAGA AAGGCGGACA GGTATCCGGT AAGCGGCAGG GTCGGAACAG
3121 GAGAGCGCAC GAGGGAGCTT CCAGGGGGAA ACGCCTGGTA TCTTTATAGT CCTGTCGGGT
3181 TTCGCCACCT CTGACTTGAG CGTCGATTTT TGTGATGCTC GTCAGGGGGG CGGAGCCTAT
3241 GGAAAAACGC CAGCAACGCG GCCTTTTTAC GGTTCCTGGC CTTTTGCTGG CCTTTTGCTC
3301 ACATGTTCTT TCCTGCGTTA TCCCCTGATT CTGTGGATAA CCGTATTACC GCCATGCATT
3361 AGTTATTAAT AGTAATCAAT TACGGGGTCA TTAGTTCATA GCCCATATAT GGAGTTCCGC
3421 GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGC CCAACGACCC CCGCCCATTG
3481 ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAG GGACTTTCCA TTGACGTCAA
3541 TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC ATCAAGTGTA TCATATGCCA
3601 AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG CCTGGCATTA TGCCCAGTAC
3661 ATGACCTTAT GGGACTTTCC TACTTGGCAG TACATCTACG TATTAGTCAT CGCTATTACC
3721 ATGGTGATGC GGTTTTGGCA GTACATCAAT GGGCGTGGAT AGCGGTTTGA CTCACGGGGA
3781 TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT TTTGGCACCA AAATCAACGG
3841 GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC AAATGGGCGG TAGGCGTGTA
3901 CGGTGGGAGG TCTATATAAG CAGAGCTGGT TTAGTGAACC GTCAGATCCG CTAGCGCTAC
3961 CGGTCGCCAC CATGGTGAGC AAGGGCGCCG AGCTGTTCAC CGGCATCGTG CCCATCCTGA
4021 TCGAGCTGAA TGGCGATGTG AATGGCCACA AGTTCAGCGT GAGCGGCGAG GGCGAGGGCG
4081 ATGCCACCTA CGGCAAGCTG ACCCTGAAGT TCATCTGCAC CACCGGCAAG CTGCCTGTGC
4141 CCTGGCCCAC CCTGGTGACC ACCCTGAGCT ACGGCGTGCA GTGCTTCTCA CGCTACCCCG
4201 ATCACATGAA GCAGCACGAC TTCTTCAAGA GCGCCATGCC TGAGGGCTAC ATCCAGGAGC
4261 GCACCATCTT CTTCGAGGAT GACGGCAACT ACAAGTCGCG CGCCGAGGTG AAGTTCGAGG
4321 GCGATACCCT GGTGAATCGC ATCGAGCTGA CCGGCACCGA TTTCAAGGAG GATGGCAACA
4381 TCCTGGGCAA TAAGATGGAG TACAACTACA ACGCCCACAA TGTGTACATC ATGACCGACA
4441 AGGCCAAGAA TGGCATCAAG GTGAACTTCA AGATCCGCCA CAACATCGAG GATGGCAGCG
4501 TGCAGCTGGC CGACCACTAC CAGCAGAATA CCCCCATCGG CGATGGCCCT GTGCTGCTGC
4561 CCGATAACCA CTACCTGTCC ACCCAGAGCG CCCTGTCCAA GGACCCCAAC GAGAAGCGCG
4621 ATCACATGAT CTACTTCGGC TTCGTGACCG CCGCCGCCAT CACCCACGGC ATGGATGAGC
4681 TGTACAAGTC CGGACTTAAG GCCTCTGTCG A
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