pACGFP1- N1 Plasmid

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pACGFP1-N1

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pACGFP1-N1,Plasmid pACGFP1-N1,pACGFP1-N1 vector

 

 

pACGFP1-N1 Information

Promoter: CMV promoter

Replicon: pUC ori, F1 ori

Terminator: SV40 poly (A) signal

Plasmid classification: lactation serial plasmids; lactating fluorescent plasmid; lactation green plasmid

Plasmid size: 4726bp

Plasmid tagging: C-GFP

Prokaryotic resistance: kanamycin Kan (50 g/ml)

Screening marker: neomycin Neo/G418

Cloning strains: E. coli DH5 and E.

Culture conditions: 37 C, aerobic LB

Expression host: mammalian cells such as 293T

Culture conditions: 37 C, 5%CO2

Induction mode: no need to induce, transient expression.

5'sequencing primers: CMV-F (CGCAAATGGGCGGTAGGCGTG)

3'sequencing primers: Sv40-polyA-R (GAAATTTGTGATGCTATTGC)

 

pACGFP1-N1 Description

 pAcGFP1-N1 encodes a green fluorescent protein (GFP) from Aequorea coerulescens (excitation maximum = 475 nm; emission maximum = 505 nm). The coding sequence of the AcGFP1 gene
contains silent base changes, which correspond to human codon-usage preferences (1). The MCS in pAcGFP1-N1 is between the immediate early promoter of CMV (PCMV IE) and the AcGFP1 coding
sequences. Genes cloned into the MCS will be expressed as fusions to the N-terminus of AcGFP1 if they are in the same reading frame as AcGFP1 and there are no intervening stop codons. SV40
polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3' end of theAcGFP1 mRNA. The vector backbone also contains an SV40 origin for replication in mammalian
cells expressing the SV40 T antigen.Aneomycin-resistance cassette (Neor), consisting of the SV40 earlypromoter,theneomycin/kanamycinresistancegeneofTn5,andpolyadenylationsignals fromthe
Herpes simplex virus thymidine kinase (HSV TK) gene, allows stably transfected eukaryotic cells to be selected using G418.Abacterial promoter upstream of the gene expresses kanamycin resistance
in E. coli. The pAcGFP1-N1 backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.
        Fusions to the N terminus of AcGFP1 retain the fluorescent properties of the native protein allowing the localization of the fusion protein in vivo . The target gene should be cloned into pAcGFP1-N1
so that it is in frame with the AcGFP1 coding sequences, with no intervening in-frame stop codons. The inserted gene should include the initiating ATG codon. The recombinant AcGFP1 vector can
be transfected into mammalian cells using any standard transfection method. If required, stable transformants can be selected using G418 (2). pAcGFP1-N1 can also be used simply to express
AcGFP1 in a cell line of interest (e.g., as a transfection marker).
• Human cytomegalovirus (CMV) immediate early promoter: 1–589
        Enhancer region:59–465; TATA box: 554–560
        Transcription start point: 583
        C→G mutation to remove Sac I site: 569
        • Multiple Cloning Site (MCS): 591–671
        • Aequorea coerulescens Green Fluorescent Protein (AcGFP): 673–1389
        Start codon (ATG): 673–675; Stop codon: 1390–1392
        Insertion of Val at position 2: 676–678
        Las amino acid: 1387–1389
• SV40 early mRNA polyadenylation signal
        Polyadenylation signals: 1545–1550 & 1574–1579; mRNA 3' ends: 1583 & 1595
• f1 single-strand DNA origin: 1642–2097 (Packages the noncoding strand of AcGFP)
• Bacterial promoter for expression of Kanr gene:
        –35 region: 2159–2164; –10 region: 2159–2164
        Transcription start point: 2154
• SV40 origin of replication: 2438–2573
• SV40 early promoter
        Enhancer (72-bp tandem repeats): 2271–2342 & 2343–2414
        21-bp repeats: 2418–2438, 2439–2459 & 2467–2481
        Early promoter element: 2494–2500
        Major transcription start points: 2490, 2528, 2534 & 2539
• Kanamycin/neomycin resistance gene:
        Neomycin phosphotransferase coding sequences: start codon (ATG): 2622–2624; stop codon: 3414–3416
        GA mutation to remove Pst I site: 2804
        C-A (Arg to Ser) mutation to remove BssHII site: 3150
• Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal
        Polyadenylation signals: 3652–3657 & 3665–3670
• pUC plasmid replication origin: 4001–4644

 

pACGFP1-N1 Multiple cloning site

pACGFP1-N1-vector

 

pACGFP1-N1 Sequence

LOCUS       Exported                4726 bp ds-DNA    circular SYN 18-10-2015
KEYWORDS    Untitled 2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4726)
  AUTHORS   admin
  TITLE     Direct Submission
  JOURNAL   Exported 2015-10-18   
FEATURES             Location/Qualifiers
     source          1..4726
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        61..364
                     /note="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /note="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     misc_feature    591..671
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             673..1392
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /note="AcGFP1"
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    1514..1635
                     /note="SV40 poly(A) signal"
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1642..2097)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     promoter        2124..2228
                     /gene="bla"
                     /note="AmpR promoter"
     promoter        2230..2587
                     /note="SV40 promoter"
                     /note="SV40 enhancer and early promoter"
     rep_origin      2438..2573
                     /note="SV40 ori"
                     /note="SV40 origin of replication"
     CDS             2622..3416
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /note="NeoR/KanR"
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    3648..3695
                     /note="HSV TK poly(A) signal"
                     /note="herpesvirus thymidine kinase polyadenylation signal"
     rep_origin      4024..4612
                     /direction=RIGHT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
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