pZsGreen1-DR

pZsGreen1-DR

pZsGreen1-DR载体基本信息：

pZsGreen1-DR载体质粒图谱和多克隆位点信息

pZsGreen1-DR载体描述：

pZsGreen1-DR is a promoterless vector that encodes ZsGreen1-DR, a destabilized variant of

Zoanthus sp. green fluorescent protein (ZsGreen; 1). The ZsGreen1 coding sequence contains

a series of silent base-pair changes, which correspond to human codon-usage preferences, for

optimal expression in mammalian cells (2). In contrast to the original protein, ZsGreen1-DR has a

short half-life, making it well suited for studies that require rapid reporter turnover. This destabilized

variant was constructed by fusing the C-terminus of the protein to amino acid residues 422–461 of

mouse ornithine decarboxylase (MODC), one of the most short-lived proteins in mammalian cells (3).

This region of MODC contains a PEST sequence that targets the protein for degradation, resulting

in rapid protein turnover (3,4). Three point mutations in this sequence further reduce the protein

half-life (3). Sequences upstream of ZsGreen1-DR have been converted to a Kozak consensus

translation initiation site (5) to enhance translation efficiency in eukaryotic cells. A single amino acid

substitution (Asn-65 to Met) has been made to enhance the emission characteristics of ZsGreen1

(excitation maximum = 496 nm; emission maximum = 506 nm).

pZsGreen1-DR can be used to monitor transcription from different promoters and promoter/enhancer

combinations inserted into the multiple cloning site (MCS), located upstream of the ZsGreen1-DR

coding sequence. Downstream SV40 polyadenylation signals direct proper processing of the 3'

end of the ZsGreen1-DR mRNA. The vector backbone contains an SV40 origin for replication in

mammalian cells expressing the SV40 large 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.

pZsGreen1-DR载体使用：

ZsGreen1-DR can be used as an in vivo reporter of gene expression. Because of its rapid turnover rate, its expression from a promoter

of interest provides a more accurate assessment of the promoter's activity over time than does the more stable ZsGreen1.

Promoter/enhancer elements should be inserted in the MCS upstream of the ZsGreen1-DR coding sequence. Without the addition of a

functional promoter, this vector will not express ZsGreen1-DR. The recombinant pZsGreen1-DR vector can be transfected into mammalian

cells using any standard transfection method. If required, stable transformants can be selected using G418 (6).

Location of features

 MCS: 12–83

 Destabilized Zoanthus sp. Green Fluorescent Protein (ZsGreen1-DR) gene

Kozak consensus translation initiation site: 83–93

Start codon (ATG): 90–92; Stop codon: 915–917

Asn-65 to Met mutation (A→T, C→G ): 286, 287

Mouse ornithine decarboxylase PEST sequence: 795–917

 SV40 early mRNA polyadenylation signal

Polyadenylation signals: 1070–1075 & 1099–1104

mRNA 3' ends: 1108 & 1120

 f1 single-strand DNA origin: 1167–1622

(Packages noncoding strand of ZsGreen1-DR.)

 Ampicillin resistance (β-lactamase) promoter

–35 region: 1684–1689; –10 region: 1707–1712

Transcription start point: 1719

 SV40 origin of replication: 1963–2098

 SV40 early promoter

Enhancer (72-bp tandem repeats): 1794–1867 & 1868–1939

21-bp repeats: 1943–1963, 1964–1984 & 1986–2006

Early promoter element: 2019–2025

Major transcription start points: 2015, 2053, 2059 & 2064

 Kanamycin/neomycin resistance geneNeomycin phosphotransferase coding sequences:

Start codon (ATG): 2147–2149; stop codon: 2939–2941

G→A mutation to remove Pst I site: 2329

C→A (Arg→Ser) mutation to remove BssH II site: 2675

 Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signal

Polyadenylation signals: 3177–3182 & 3190–3195

 pUC plasmid replication origin: 3526–4169

Excitation and emission maxima of ZsGreen1

 Excitation maximum = 496 nm

 Emission maximum = 506 nm

pZsGreen1-DR载体序列：

ORIGIN

    1 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG

   61 ACGGTACCGC GGGCCCGGGA TCCGCCACCA TGGCCCAGTC CAAGCACGGC CTGACCAAGG

  121 AGATGACCAT GAAGTACCGC ATGGAGGGCT GCGTGGACGG CCACAAGTTC GTGATCACCG

  181 GCGAGGGCAT CGGCTACCCC TTCAAGGGCA AGCAGGCCAT CAACCTGTGC GTGGTGGAGG

  241 GCGGCCCCTT GCCCTTCGCC GAGGACATCT TGTCCGCCGC CTTCATGTAC GGCAACCGCG

  301 TGTTCACCGA GTACCCCCAG GACATCGTCG ACTACTTCAA GAACTCCTGC CCCGCCGGCT

  361 ACACCTGGGA CCGCTCCTTC CTGTTCGAGG ACGGCGCCGT GTGCATCTGC AACGCCGACA

  421 TCACCGTGAG CGTGGAGGAG AACTGCATGT ACCACGAGTC CAAGTTCTAC GGCGTGAACT

  481 TCCCCGCCGA CGGCCCCGTG ATGAAGAAGA TGACCGACAA CTGGGAGCCC TCCTGCGAGA

  541 AGATCATCCC CGTGCCCAAG CAGGGCATCT TGAAGGGCGA CGTGAGCATG TACCTGCTGC

  601 TGAAGGACGG TGGCCGCTTG CGCTGCCAGT TCGACACCGT GTACAAGGCC AAGTCCGTGC

  661 CCCGCAAGAT GCCCGACTGG CACTTCATCC AGCACAAGCT GACCCGCGAG GACCGCAGCG

  721 ACGCCAAGAA CCAGAAGTGG CACCTGACCG AGCACGCCAT CGCCTCCGGC TCCGCCTTGC

  781 CCAAGCTTCC GCGGAGCCAT GGCTTCCCGC CGGCGGTGGC GGCGCAGGAT GATGGCACGC

  841 TGCCCATGTC TTGTGCCCAG GAGAGCGGGA TGGACCGTCA CCCTGCAGCC TGTGCTTCTG

  901 CTAGGATCAA TGTGTAGGCG GCCGCGACTC TAGATCATAA TCAGCCATAC CACATTTGTA

  961 GAGGTTTTAC TTGCTTTAAA AAACCTCCCA CACCTCCCCC TGAACCTGAA ACATAAAATG

 1021 AATGCAATTG TTGTTGTTAA CTTGTTTATT GCAGCTTATA ATGGTTACAA ATAAAGCAAT

 1081 AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC ATTCTAGTTG TGGTTTGTCC

 1141 AAACTCATCA ATGTATCTTA AGGCGTAAAT TGTAAGCGTT AATATTTTGT TAAAATTCGC

 1201 GTTAAATTTT TGTTAAATCA GCTCATTTTT TAACCAATAG GCCGAAATCG GCAAAATCCC

 1261 TTATAAATCA AAAGAATAGA CCGAGATAGG GTTGAGTGTT GTTCCAGTTT GGAACAAGAG

 1321 TCCACTATTA AAGAACGTGG ACTCCAACGT CAAAGGGCGA AAAACCGTCT ATCAGGGCGA

 1381 TGGCCCACTA CGTGAACCAT CACCCTAATC AAGTTTTTTG GGGTCGAGGT GCCGTAAAGC

 1441 ACTAAATCGG AACCCTAAAG GGAGCCCCCG ATTTAGAGCT TGACGGGGAA AGCCGGCGAA

 1501 CGTGGCGAGA AAGGAAGGGA AGAAAGCGAA AGGAGCGGGC GCTAGGGCGC TGGCAAGTGT

 1561 AGCGGTCACG CTGCGCGTAA CCACCACACC CGCCGCGCTT AATGCGCCGC TACAGGGCGC

 1621 GTCAGGTGGC ACTTTTCGGG GAAATGTGCG CGGAACCCCT ATTTGTTTAT TTTTCTAAAT

 1681 ACATTCAAAT ATGTATCCGC TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG

 1741 AAAAAGGAAG AGTCCTGAGG CGGAAAGAAC CAGCTGTGGA ATGTGTGTCA GTTAGGGTGT

 1801 GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA AGTATGCAAA GCATGCATCT CAATTAGTCA

 1861 GCAACCAGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA GAAGTATGCA AAGCATGCAT

 1921 CTCAATTAGT CAGCAACCAT AGTCCCGCCC CTAACTCCGC CCATCCCGCC CCTAACTCCG

 1981 CCCAGTTCCG CCCATTCTCC GCCCCATGGC TGACTAATTT TTTTTATTTA TGCAGAGGCC

 2041 GAGGCCGCCT CGGCCTCTGA GCTATTCCAG AAGTAGTGAG GAGGCTTTTT TGGAGGCCTA

 2101 GGCTTTTGCA AAGATCGATC AAGAGACAGG ATGAGGATCG TTTCGCATGA TTGAACAAGA

 2161 TGGATTGCAC GCAGGTTCTC CGGCCGCTTG GGTGGAGAGG CTATTCGGCT ATGACTGGGC

 2221 ACAACAGACA ATCGGCTGCT CTGATGCCGC CGTGTTCCGG CTGTCAGCGC AGGGGCGCCC

 2281 GGTTCTTTTT GTCAAGACCG ACCTGTCCGG TGCCCTGAAT GAACTGCAAG ACGAGGCAGC

 2341 GCGGCTATCG TGGCTGGCCA CGACGGGCGT TCCTTGCGCA GCTGTGCTCG ACGTTGTCAC

 2401 TGAAGCGGGA AGGGACTGGC TGCTATTGGG CGAAGTGCCG GGGCAGGATC TCCTGTCATC

 2461 TCACCTTGCT CCTGCCGAGA AAGTATCCAT CATGGCTGAT GCAATGCGGC GGCTGCATAC

 2521 GCTTGATCCG GCTACCTGCC CATTCGACCA CCAAGCGAAA CATCGCATCG AGCGAGCACG

 2581 TACTCGGATG GAAGCCGGTC TTGTCGATCA GGATGATCTG GACGAAGAGC ATCAGGGGCT

 2641 CGCGCCAGCC GAACTGTTCG CCAGGCTCAA GGCGAGCATG CCCGACGGCG AGGATCTCGT

 2701 CGTGACCCAT GGCGATGCCT GCTTGCCGAA TATCATGGTG GAAAATGGCC GCTTTTCTGG

 2761 ATTCATCGAC TGTGGCCGGC TGGGTGTGGC GGACCGCTAT CAGGACATAG CGTTGGCTAC

 2821 CCGTGATATT GCTGAAGAGC TTGGCGGCGA ATGGGCTGAC CGCTTCCTCG TGCTTTACGG

 2881 TATCGCCGCT CCCGATTCGC AGCGCATCGC CTTCTATCGC CTTCTTGACG AGTTCTTCTG

 2941 AGCGGGACTC TGGGGTTCGA AATGACCGAC CAAGCGACGC CCAACCTGCC ATCACGAGAT

 3001 TTCGATTCCA CCGCCGCCTT CTATGAAAGG TTGGGCTTCG GAATCGTTTT CCGGGACGCC

 3061 GGCTGGATGA TCCTCCAGCG CGGGGATCTC ATGCTGGAGT TCTTCGCCCA CCCTAGGGGG

 3121 AGGCTAACTG AAACACGGAA GGAGACAATA CCGGAAGGAA CCCGCGCTAT GACGGCAATA

 3181 AAAAGACAGA ATAAAACGCA CGGTGTTGGG TCGTTTGTTC ATAAACGCGG GGTTCGGTCC

 3241 CAGGGCTGGC ACTCTGTCGA TACCCCACCG AGACCCCATT GGGGCCAATA CGCCCGCGTT

 3301 TCTTCCTTTT CCCCACCCCA CCCCCCAAGT TCGGGTGAAG GCCCAGGGCT CGCAGCCAAC

 3361 GTCGGGGCGG CAGGCCCTGC CATAGCCTCA GGTTACTCAT ATATACTTTA GATTGATTTA

 3421 AAACTTCATT TTTAATTTAA AAGGATCTAG GTGAAGATCC TTTTTGATAA TCTCATGACC

 3481 AAAATCCCTT AACGTGAGTT TTCGTTCCAC TGAGCGTCAG ACCCCGTAGA AAAGATCAAA

 3541 GGATCTTCTT GAGATCCTTT TTTTCTGCGC GTAATCTGCT GCTTGCAAAC AAAAAAACCA

 3601 CCGCTACCAG CGGTGGTTTG TTTGCCGGAT CAAGAGCTAC CAACTCTTTT TCCGAAGGTA

 3661 ACTGGCTTCA GCAGAGCGCA GATACCAAAT ACTGTCCTTC TAGTGTAGCC GTAGTTAGGC

 3721 CACCACTTCA AGAACTCTGT AGCACCGCCT ACATACCTCG CTCTGCTAAT CCTGTTACCA

 3781 GTGGCTGCTG CCAGTGGCGA TAAGTCGTGT CTTACCGGGT TGGACTCAAG ACGATAGTTA

 3841 CCGGATAAGG CGCAGCGGTC GGGCTGAACG GGGGGTTCGT GCACACAGCC CAGCTTGGAG

 3901 CGAACGACCT ACACCGAACT GAGATACCTA CAGCGTGAGC TATGAGAAAG CGCCACGCTT

 3961 CCCGAAGGGA GAAAGGCGGA CAGGTATCCG GTAAGCGGCA GGGTCGGAAC AGGAGAGCGC

 4021 ACGAGGGAGC TTCCAGGGGG AAACGCCTGG TATCTTTATA GTCCTGTCGG GTTTCGCCAC

 4081 CTCTGACTTG AGCGTCGATT TTTGTGATGC TCGTCAGGGG GGCGGAGCCT ATGGAAAAAC

 4141 GCCAGCAACG CGGCCTTTTT ACGGTTCCTG GCCTTTTGCT GGCCTTTTGC TCACATGTTC

 4201 TTTCCTGCGT TATCCCCTGA TTCTGTGGAT AACCGTATTA CCGCCATGCA T

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