pZsGreen1-1

pZsGreen1-1

pZsGreen1-1载体基本信息：

pZsGreen1-1载体质粒图谱和多克隆位点信息

pZsGreen1-1载体简介：

pZsGreen1-1 encodes a human codon-optimized variant of wild-type Zoanthus sp. green fluorescent

protein, ZsGreen1 (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). A single amino acid substitution (Asn-66 to Met) has been made to enhance the emission

characteristics of ZsGreen1 (excitation maximum = 493 nm; emission maximum = 505 nm).

pZsGreen1-1 is a promoterless ZsGreen1 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 ZsGreen1 have been converted to a Kozak consensus translation

initiation site (3) to increase translation efficiency in eukaryotic cells. SV40 polyadenylation signals

downstream of the ZsGreen1 gene direct proper processing of the 3' end of the ZsGreen1 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 singlestranded

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.

pZsGreen1-1载体使用：

ZsGreen1 can be used as an in vivo reporter of gene expression. Promoters should be cloned

into the pZsGreen1-1 MCS upstream from the ZsGreen1 coding sequence. Without addition of a

functional promoter, this vector will not express ZsGreen1. The recombinant ZsGreen1 vector can

be transfected into mammalian cells using any standard transfection method. If required, stable

transfectants can be selected using G418 (4).

pZsGreen1-1载体特征：

 MCS: 12–83

 Zoanthus sp. human codon-optimized Green Fluorescent Protein (ZsGreen1) gene

Kozak consensus translation initiation site: 90–100

Start codon (ATG): 97–99; Stop codon: 790–792

Asn-66 to Met mutation (A→T, C→G ): 293, 294

 SV40 early mRNA polyadenylation signal

Polyadenylation signals: 945–950 & 974–979

mRNA 3' ends: 983 & 995

 f1 single-strand DNA origin: 1042–1497

(Packages noncoding strand of ZsGreen1.)

 Ampicillin resistance (β-lactamase) promoter

–35 region: 1559–1564; –10 region: 1582–1587

Transcription start point: 1594

 SV40 origin of replication: 1838–1973

 SV40 early promoter

Enhancer (72-bp tandem repeats): 1669–1742 & 1743–1814

21-bp repeats: 1818–1837, 1839–1859 & 1861–1881

Early promoter element: 1894–1900

Major transcription start points: 1890, 1928, 1934 & 1939

 Kanamycin/neomycin resistance geneNeomycin phosphotransferase coding sequences:

Start codon (ATG): 2022–2024; stop codon: 2814–2816

G→A mutation to remove Pst I site: 2204

C→A (Arg→Ser) mutation to remove BssH II site: 2550

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

Polyadenylation signals: 3052–3057 & 3065–3070

 pUC plasmid replication origin: 3401–4044

Propagation in E. Coli

 Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires

a host containing an F plasmid such as JM109 or XL1-Blue.

 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/Col E1

Green Fluorescent Protein (ZsGreen1)

 Excitation/Emission Maxima: 493 nm / 505 nm

pZsGreen1-1载体序列：

ORIGIN

    1 TAGTTATTAC TAGCGCTACC GGACTCAGAT CTCGAGCTCA AGCTTCGAAT TCTGCAGTCG

   61 ACGGTACCGC GGGCCCGGGA TCCACCGGTC GCCACCATGG CCCAGTCCAA GCACGGCCTG

  121 ACCAAGGAGA TGACCATGAA GTACCGCATG GAGGGCTGCG TGGACGGCCA CAAGTTCGTG

  181 ATCACCGGCG AGGGCATCGG CTACCCCTTC AAGGGCAAGC AGGCCATCAA CCTGTGCGTG

  241 GTGGAGGGCG GCCCCTTGCC CTTCGCCGAG GACATCTTGT CCGCCGCCTT CATGTACGGC

  301 AACCGCGTGT TCACCGAGTA CCCCCAGGAC ATCGTCGACT ACTTCAAGAA CTCCTGCCCC

  361 GCCGGCTACA CCTGGGACCG CTCCTTCCTG TTCGAGGACG GCGCCGTGTG CATCTGCAAC

  421 GCCGACATCA CCGTGAGCGT GGAGGAGAAC TGCATGTACC ACGAGTCCAA GTTCTACGGC

  481 GTGAACTTCC CCGCCGACGG CCCCGTGATG AAGAAGATGA CCGACAACTG GGAGCCCTCC

  541 TGCGAGAAGA TCATCCCCGT GCCCAAGCAG GGCATCTTGA AGGGCGACGT GAGCATGTAC

  601 CTGCTGCTGA AGGACGGTGG CCGCTTGCGC TGCCAGTTCG ACACCGTGTA CAAGGCCAAG

  661 TCCGTGCCCC GCAAGATGCC CGACTGGCAC TTCATCCAGC ACAAGCTGAC CCGCGAGGAC

  721 CGCAGCGACG CCAAGAACCA GAAGTGGCAC CTGACCGAGC ACGCCATCGC CTCCGGCTCC

  781 GCCTTGCCCT GAGCGGCCGC GACTCTAGAT CATAATCAGC CATACCACAT TTGTAGAGGT

  841 TTTACTTGCT TTAAAAAACC TCCCACACCT CCCCCTGAAC CTGAAACATA AAATGAATGC

  901 AATTGTTGTT GTTAACTTGT TTATTGCAGC TTATAATGGT TACAAATAAA GCAATAGCAT

  961 CACAAATTTC ACAAATAAAG CATTTTTTTC ACTGCATTCT AGTTGTGGTT TGTCCAAACT

 1021 CATCAATGTA TCTTAAGGCG TAAATTGTAA GCGTTAATAT TTTGTTAAAA TTCGCGTTAA

 1081 ATTTTTGTTA AATCAGCTCA TTTTTTAACC AATAGGCCGA AATCGGCAAA ATCCCTTATA

 1141 AATCAAAAGA ATAGACCGAG ATAGGGTTGA GTGTTGTTCC AGTTTGGAAC AAGAGTCCAC

 1201 TATTAAAGAA CGTGGACTCC AACGTCAAAG GGCGAAAAAC CGTCTATCAG GGCGATGGCC

 1261 CACTACGTGA ACCATCACCC TAATCAAGTT TTTTGGGGTC GAGGTGCCGT AAAGCACTAA

 1321 ATCGGAACCC TAAAGGGAGC CCCCGATTTA GAGCTTGACG GGGAAAGCCG GCGAACGTGG

 1381 CGAGAAAGGA AGGGAAGAAA GCGAAAGGAG CGGGCGCTAG GGCGCTGGCA AGTGTAGCGG

 1441 TCACGCTGCG CGTAACCACC ACACCCGCCG CGCTTAATGC GCCGCTACAG GGCGCGTCAG

 1501 GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC TAAATACATT

 1561 CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT GCTTCAATAA TATTGAAAAA

 1621 GGAAGAGTCC TGAGGCGGAA AGAACCAGCT GTGGAATGTG TGTCAGTTAG GGTGTGGAAA

 1681 GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC

 1741 CAGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT ATGCAAAGCA TGCATCTCAA

 1801 TTAGTCAGCA ACCATAGTCC CGCCCCTAAC TCCGCCCATC CCGCCCCTAA CTCCGCCCAG

 1861 TTCCGCCCAT TCTCCGCCCC ATGGCTGACT AATTTTTTTT ATTTATGCAG AGGCCGAGGC

 1921 CGCCTCGGCC TCTGAGCTAT TCCAGAAGTA GTGAGGAGGC TTTTTTGGAG GCCTAGGCTT

 1981 TTGCAAAGAT CGATCAAGAG ACAGGATGAG GATCGTTTCG CATGATTGAA CAAGATGGAT

 2041 TGCACGCAGG TTCTCCGGCC GCTTGGGTGG AGAGGCTATT CGGCTATGAC TGGGCACAAC

 2101 AGACAATCGG CTGCTCTGAT GCCGCCGTGT TCCGGCTGTC AGCGCAGGGG CGCCCGGTTC

 2161 TTTTTGTCAA GACCGACCTG TCCGGTGCCC TGAATGAACT GCAAGACGAG GCAGCGCGGC

 2221 TATCGTGGCT GGCCACGACG GGCGTTCCTT GCGCAGCTGT GCTCGACGTT GTCACTGAAG

 2281 CGGGAAGGGA CTGGCTGCTA TTGGGCGAAG TGCCGGGGCA GGATCTCCTG TCATCTCACC

 2341 TTGCTCCTGC CGAGAAAGTA TCCATCATGG CTGATGCAAT GCGGCGGCTG CATACGCTTG

 2401 ATCCGGCTAC CTGCCCATTC GACCACCAAG CGAAACATCG CATCGAGCGA GCACGTACTC

 2461 GGATGGAAGC CGGTCTTGTC GATCAGGATG ATCTGGACGA AGAGCATCAG GGGCTCGCGC

 2521 CAGCCGAACT GTTCGCCAGG CTCAAGGCGA GCATGCCCGA CGGCGAGGAT CTCGTCGTGA

 2581 CCCATGGCGA TGCCTGCTTG CCGAATATCA TGGTGGAAAA TGGCCGCTTT TCTGGATTCA

 2641 TCGACTGTGG CCGGCTGGGT GTGGCGGACC GCTATCAGGA CATAGCGTTG GCTACCCGTG

 2701 ATATTGCTGA AGAGCTTGGC GGCGAATGGG CTGACCGCTT CCTCGTGCTT TACGGTATCG

 2761 CCGCTCCCGA TTCGCAGCGC ATCGCCTTCT ATCGCCTTCT TGACGAGTTC TTCTGAGCGG

 2821 GACTCTGGGG TTCGAAATGA CCGACCAAGC GACGCCCAAC CTGCCATCAC GAGATTTCGA

 2881 TTCCACCGCC GCCTTCTATG AAAGGTTGGG CTTCGGAATC GTTTTCCGGG ACGCCGGCTG

 2941 GATGATCCTC CAGCGCGGGG ATCTCATGCT GGAGTTCTTC GCCCACCCTA GGGGGAGGCT

 3001 AACTGAAACA CGGAAGGAGA CAATACCGGA AGGAACCCGC GCTATGACGG CAATAAAAAG

 3061 ACAGAATAAA ACGCACGGTG TTGGGTCGTT TGTTCATAAA CGCGGGGTTC GGTCCCAGGG

 3121 CTGGCACTCT GTCGATACCC CACCGAGACC CCATTGGGGC CAATACGCCC GCGTTTCTTC

 3181 CTTTTCCCCA CCCCACCCCC CAAGTTCGGG TGAAGGCCCA GGGCTCGCAG CCAACGTCGG

 3241 GGCGGCAGGC CCTGCCATAG CCTCAGGTTA CTCATATATA CTTTAGATTG ATTTAAAACT

 3301 TCATTTTTAA TTTAAAAGGA TCTAGGTGAA GATCCTTTTT GATAATCTCA TGACCAAAAT

 3361 CCCTTAACGT GAGTTTTCGT TCCACTGAGC GTCAGACCCC GTAGAAAAGA TCAAAGGATC

 3421 TTCTTGAGAT CCTTTTTTTC TGCGCGTAAT CTGCTGCTTG CAAACAAAAA AACCACCGCT

 3481 ACCAGCGGTG GTTTGTTTGC CGGATCAAGA GCTACCAACT CTTTTTCCGA AGGTAACTGG

 3541 CTTCAGCAGA GCGCAGATAC CAAATACTGT TCTTCTAGTG TAGCCGTAGT TAGGCCACCA

 3601 CTTCAAGAAC TCTGTAGCAC CGCCTACATA CCTCGCTCTG CTAATCCTGT TACCAGTGGC

 3661 TGCTGCCAGT GGCGATAAGT CGTGTCTTAC CGGGTTGGAC TCAAGACGAT AGTTACCGGA

 3721 TAAGGCGCAG CGGTCGGGCT GAACGGGGGG TTCGTGCACA CAGCCCAGCT TGGAGCGAAC

 3781 GACCTACACC GAACTGAGAT ACCTACAGCG TGAGCTATGA GAAAGCGCCA CGCTTCCCGA

 3841 AGGGAGAAAG GCGGACAGGT ATCCGGTAAG CGGCAGGGTC GGAACAGGAG AGCGCACGAG

 3901 GGAGCTTCCA GGGGGAAACG CCTGGTATCT TTATAGTCCT GTCGGGTTTC GCCACCTCTG

 3961 ACTTGAGCGT CGATTTTTGT GATGCTCGTC AGGGGGGCGG AGCCTATGGA AAAACGCCAG

 4021 CAACGCGGCC TTTTTACGGT TCCTGGCCTT TTGCTGGCCT TTTGCTCACA TGTTCTTTCC

 4081 TGCGTTATCC CCTGATTCTG TGGATAACCG TATTACCGCC ATGCAT

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