pZsGreen1-1载体pZsGreen1-1 encodes a human codon-optimized variant of wild-type Zoanthus sp. green fluorescentprotein, 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 emissioncharacteristics of ZsGreen1 (excitation maximum = 493 nm; emission maximum = 505 nm).pZsGreen1-1 is a promoterless ZsGreen1 vector that can be used to monitor transcription fromdifferent promoters and promoter/enhancer combinations inserted into the multiple cloning site(MCS). Sequences upstream of ZsGreen1 have been converted to a Kozak consensus translationinitiation site (3) to increase translation efficiency in eukaryotic cells. SV40 polyadenylation signalsdownstream 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 theSV40 T antigen, a pUC origin of replication for propagation in E. coli, and an f1 origin for singlestrandedDNA production. A neomycin-resistance cassette (Neor) allows stably transfectedeukaryotic cells to be selected using G418. This cassette consists of the SV40 early promoter, theneomycin/kanamycin resistance gene of Tn5, and polyadenylation signals from the Herpes simplexvirus thymidine kinase (HSV TK) gene. A bacterial promoter upstream of the cassette expresseskanamycin resistance in E. coli.载体使用:ZsGreen1 can be used as an in vivo reporter of gene expression. Promoters should be clonedinto the pZsGreen1-1 MCS upstream from the ZsGreen1 coding sequence. Without addition of afunctional promoter, this vector will not express ZsGreen1. The recombinant ZsGreen1 vector canbe transfected into mammalian cells using any standard transfection method. If required, stabletransfectants can be selected using G418 (4).载体特征: MCS: 12–83 Zoanthus sp. human codon-optimized Green Fluorescent Protein (ZsGreen1) geneKozak consensus translation initiation site: 90–100Start codon (ATG): 97–99; Stop codon: 790–792Asn-66 to Met mutation (A→T, C→G ): 293, 294 SV40 early mRNA polyadenylation signalPolyadenylation signals: 945–950 & 974–979mRNA 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: 1582–1587Transcription start point: 1594 SV40 origin of replication: 1838–1973 SV40 early promoterEnhancer (72-bp tandem repeats): 1669–1742 & 1743–181421-bp repeats: 1818–1837, 1839–1859 & 1861–1881Early promoter element: 1894–1900Major transcription start points: 1890, 1928, 1934 & 1939 Kanamycin/neomycin resistance geneNeomycin phosphotransferase coding sequences:Start codon (ATG): 2022–2024; stop codon: 2814–2816G→A mutation to remove Pst I site: 2204C→A (Arg→Ser) mutation to remove BssH II site: 2550 Herpes simplex virus (HSV) thymidine kinase (TK) polyadenylation signalPolyadenylation signals: 3052–3057 & 3065–3070 pUC plasmid replication origin: 3401–4044Propagation in E. Coli Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requiresa 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 E1Green Fluorescent Protein (ZsGreen1) Excitation/Emission Maxima: 493 nm / 505 nm
LOCUS pZsGreen1-1 4126 bp DNA SYNDEFINITION pZsGreen1-1ACCESSION KEYWORDS SOURCE ORGANISM other sequences; artificial sequences; vectors.FEATURES Location/Qualifiers source 1..4126 /organism="pZsGreen1-1" /mol_type="other DNA" CDS 97..792 /label="ORF frame 1" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAI NLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDG AVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGI LKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWH LTEHAIASGSALP*" gene 100..789 /label="Zs_Green" /gene="Zs_Green" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAI NLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDG AVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGI LKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWH LTEHAIASGSALP*" misc_feature 1005..1024 /label="EBV_rev_primer" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAI NLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDG AVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGI LKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWH LTEHAIASGSALP*" rep_origin complement(1174..1480) /label="f1_origin" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAI NLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDG AVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGI LKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWH LTEHAIASGSALP*" promoter 1559..1587 /label="AmpR_promoter" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAI NLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDG AVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGI LKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWH LTEHAIASGSALP*" misc_feature complement(1653..1673) /label="pBABE_3_primer" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAI NLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDG AVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGI LKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWH LTEHAIASGSALP*" misc_feature complement(1659..1874) /label="SV40_enhancer" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAI NLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDG AVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGI LKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWH LTEHAIASGSALP*" promoter 1671..1939 /label="SV40_promoter" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAI NLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDG AVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGI LKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWH LTEHAIASGSALP*" rep_origin 1838..1915 /label="SV40_origin" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAI NLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDG AVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGI LKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWH LTEHAIASGSALP*" misc_feature 1900..1919 /label="SV40pro_F_primer" /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAI NLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDG AVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGI LKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWH LTEHAIASGSALP*" CDS 2022..2816 /label="ORF frame 3" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" gene 2025..2813 /label="NeoR/KanR" /gene="NeoR/KanR" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*" CDS complement(2331..2867) /label="ORF frame 3" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" terminator 2991..3260 /label="TK_PA_terminator" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*" rep_origin 3408..4027 /label="pBR322_origin" /translation="MAGWASLGRSFRTPESRSEELVKKAIEGDALRIGSGDTVKHEEA VSPFAAKLFSNITGSQRYVLIAVRHTQPATVDESRKAAIFHHDIRQAGIAMGHDEILA VGHARLEPGEQFGWREPLMLFVQIILIDKTGFHPSTCSLDAMFRLVVEWAGSRIKRMQ PPHCISHDGYFLGRSKVR*"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//