pACGFP1- N1 Plasmid

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  • Model: PVT1211
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pACGFP1-N1

PVT1211       2ug
 

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

      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 ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc gcgggcccgg

      661 gatccaccgg tcatggtgag caagggcgcc gagctgttca ccggcatcgt gcccatcctg

      721 atcgagctga atggcgatgt gaatggccac aagttcagcg tgagcggcga gggcgagggc

      781 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcctgtg

      841 ccctggccca ccctggtgac caccctgagc tacggcgtgc agtgcttctc acgctacccc

      901 gatcacatga agcagcacga cttcttcaag agcgccatgc ctgagggcta catccaggag

      961 cgcaccatct tcttcgagga tgacggcaac tacaagtcgc gcgccgaggt gaagttcgag

     1021 ggcgataccc tggtgaatcg catcgagctg accggcaccg atttcaagga ggatggcaac

     1081 atcctgggca ataagatgga gtacaactac aacgcccaca atgtgtacat catgaccgac

     1141 aaggccaaga atggcatcaa ggtgaacttc aagatccgcc acaacatcga ggatggcagc

     1201 gtgcagctgg ccgaccacta ccagcagaat acccccatcg gcgatggccc tgtgctgctg

     1261 cccgataacc actacctgtc cacccagagc gccctgtcca aggaccccaa cgagaagcgc

     1321 gatcacatga tctacttcgg cttcgtgacc gccgccgcca tcacccacgg catggatgag

     1381 ctgtacaagt gagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt

     1441 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc

     1501 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat

     1561 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact

     1621 catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa

     1681 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata

     1741 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac

     1801 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc

     1861 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa

     1921 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg

     1981 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg

     2041 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag

     2101 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt

     2161 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa

     2221 ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa

     2281 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac

     2341 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa

     2401 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag

     2461 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc

     2521 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt

     2581 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat

     2641 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac

     2701 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc

     2761 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc

     2821 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag

     2881 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc

     2941 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg

     3001 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc

     3061 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc

     3121 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga

     3181 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca

     3241 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg

     3301 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg

     3361 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg

     3421 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga

     3481 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg

     3541 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct

     3601 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag

     3661 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg

     3721 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc

     3781 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg

     3841 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact

     3901 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat

     3961 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc

     4021 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct

     4081 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg

     4141 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca

     4201 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc

     4261 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga

     4321 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac

     4381 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga

     4441 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag

     4501 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg

     4561 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag

     4621 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc

     4681 tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat

//

 

Caution:
Product is for research use only!

 

 

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

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