pAcGFP1-1
Search name
pAcGFP1-1,Plasmid pAcGFP1-1,pAcGFP1-1 vector
pAcGFP1-1 Informaiton
Replicon: pUC ori, F1 ori
Terminator: SV40 poly (A) signal
Plasmid classification: lactation serial plasmids; lactating fluorescent plasmid; lactation green plasmid
Plasmid size: 4150bp
Plasmid tagging: C-AcGFP1
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
Induction mode: no need to induce, transient expression.
5'sequencing primers: AcGFP-1-F (GCGTCGATTTTTGTGATGCTC)
3'sequencing primers: Sv40-polyA-R (GAAATTTGTGATGCTATTGC)
Remarks: mammalian cell promoter detection vector
pAcGFP1-1 Description
pAcGFP1-1 encodes the green fuorescent protein AcGFP1, a derivative of AcGFP from Aequorea coerulescens. AcGFP1 has been optimized for brighter fuorescence. (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 . pAcGFP1-1 is a promoterless vector that can be used to monitor transcription from different promoters and promoter/enhancer combinations inserted into the multiple cloning site (MCS). Sequences upstream of AcGFP1 have been converted to a Kozak consensus translation initiation site (2) to enhance translation effciency in eukaryotic cells. SV40 polyadenylation signals downstream of the AcGFP1 gene direct proper processing of the 3' end of the AcGFP1 mRNA. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for single-stranded DNA production. A neomycin-resistance cassette (Neor) allows stably transfected eukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, the neomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette expresses kanamycin resistance in E. coli.
pAcGFP1-1 Multiple cloning site
pAcGFP1-1 Sequence
LOCUS Exported 4150 bp ds-DNA circular SYN 18-10-2015
KEYWORDS Untitled
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4150)
AUTHORS admin
TITLE Direct Submission
JOURNAL Exported 2015-10-18
FEATURES Location/Qualifiers
source 1..4150
/organism="synthetic DNA construct"
/mol_type="other DNA"
misc_feature 12..89
/note="MCS"
/note="multiple cloning site"
CDS 97..816
/codon_start=1
/product="Aequorea coerulescens GFP"
/note="AcGFP1"
/note="mammalian codon-optimized"
/translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
GFVTAAAITHGMDELYK"
polyA_signal 938..1059
/note="SV40 poly(A) signal"
/note="SV40 polyadenylation signal"
rep_origin complement(1066..1521)
/direction=LEFT
/note="f1 ori"
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1548..1652
/gene="bla"
/note="AmpR promoter"
promoter 1654..2011
/note="SV40 promoter"
/note="SV40 enhancer and early promoter"
rep_origin 1862..1997
/note="SV40 ori"
/note="SV40 origin of replication"
CDS 2046..2840
/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 3072..3119
/note="HSV TK poly(A) signal"
/note="herpesvirus thymidine kinase polyadenylation signal"
rep_origin 3448..4036
/direction=RIGHT
/note="ori"
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
ORIGIN
1 tagttattac tagcgctacc ggactcagat ctcgagctca agcttcgaat tctgcagtcg
61 acggtaccgc gggcccggga tccaccggtc gccaccatgg tgagcaaggg cgccgagctg
121 ttcaccggca tcgtgcccat cctgatcgag ctgaatggcg atgtgaatgg ccacaagttc
181 agcgtgagcg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
241 tgcaccaccg gcaagctgcc tgtgccctgg cccaccctgg tgaccaccct gagctacggc
301 gtgcagtgct tctcacgcta ccccgatcac atgaagcagc acgacttctt caagagcgcc
361 atgcctgagg gctacatcca ggagcgcacc atcttcttcg aggatgacgg caactacaag
421 tcgcgcgccg aggtgaagtt cgagggcgat accctggtga atcgcatcga gctgaccggc
481 accgatttca aggaggatgg caacatcctg ggcaataaga tggagtacaa ctacaacgcc
541 cacaatgtgt acatcatgac cgacaaggcc aagaatggca tcaaggtgaa cttcaagatc
601 cgccacaaca tcgaggatgg cagcgtgcag ctggccgacc actaccagca gaataccccc
661 atcggcgatg gccctgtgct gctgcccgat aaccactacc tgtccaccca gagcgccctg
721 tccaaggacc ccaacgagaa gcgcgatcac atgatctact tcggcttcgt gaccgccgcc
781 gccatcaccc acggcatgga tgagctgtac aagtgagcgg ccgcgactct agatcataat
841 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
901 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
961 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
1021 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
1081 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
1141 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
1201 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
1261 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
1321 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
1381 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
1441 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
1501 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
1561 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
1621 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
1681 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
1741 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
1801 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
1861 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
1921 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
1981 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
2041 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
2101 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
2161 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
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