LOCUS       sequence-357755-        6876 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian Expression of Fluc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6876)
  AUTHORS   Kuzuoglu-Ozturk D, Bhandari D, Huntzinger E, Fauser M, Helms S, 
            Izaurralde E
  TITLE     miRISC and the CCR4-NOT complex silence mRNA targets independently 
            of 43S ribosomal scanning.
  JOURNAL   EMBO J. 2016 Jun 1;35(11):1186-203. doi: 10.15252/embj.201592901. 
            Epub 2016 Mar 23.
  PUBMED    27009120
REFERENCE   2  (bases 1 to 6876)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.15252/embj.201592901"; journalName: "EMBO J"; date: "2016-06-1- 
            1"; volume: "35"; issue: "11"; pages: "1186-203"
COMMENT     Sequence Label: pT7-EGFP-C1-HCV-Fluc_AC
FEATURES             Location/Qualifiers
     source          1..6876
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     553..572
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        553..571
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      599..608
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(605..607,608..610,611..1321)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(605..607,608..610,611..1321)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(650..671)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(911..930)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1258..1279
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1791..3443
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(1857..1875)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     polyA_signal    3604..3725
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3641..3660)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3695..3714
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3732..4187)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3869..3890)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4081..4100
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4214..4318
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4320..4677
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4320..4511
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4343..4363)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4528..4663
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4712..5506
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4712..5506
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4766..4785)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5376..5395
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5694..5713)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5738..5785
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6114..6702
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6603..6622
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg
      541 gcttatcgaa attaatacga ctcactatag ggagacccaa gctctagcgc taccggtcgc
      601 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      661 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      721 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
      781 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
      841 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      901 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
      961 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1021 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     1081 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1141 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1201 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     1261 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1321 gtccggactc agatctcgag aataattcta gtgtacgtag ccagcccccg attgggggcg
     1381 acactccacc atagatcact cccctgtgag gaactactgt cttcacgcag aaagcgtcta
     1441 gccatggcgt tagtatgaga gtcgtgcagc ctccaggacc ccccctcccg ggagagccat
     1501 agtggtctgc ggaaccggtg agtacaccgg aattgccagg acgaccgggt cctttcttgg
     1561 atcaacccgc tcaatgcctg gagatttggg cgtgcccccg caagactgct agccgagtag
     1621 tgttgggtcg cgaaaggcct tgtggtactg cctgataggg tgcttgcgag tgccccggga
     1681 ggtctcgtag accgtgcacc atgagcacga atcctaaacc tcaaagaaaa accaaacgta
     1741 acaccaaccg ccgcccacag gacgtcttca tatgtctagc caccaagctt atggaagacg
     1801 ccaaaaacat aaagaaaggc ccggcgccat tctatccgct ggaagatgga accgctggag
     1861 agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt gcttttacag
     1921 atgcacatat cgaggtggac atcacttacg ctgagtactt cgaaatgtcc gttcggttgg
     1981 cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta tgcagtgaaa
     2041 actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc
     2101 ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgggcatt tcgcagccta
     2161 ccgtggtgtt cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa aaaaagctcc
     2221 caatcatcca aaaaattatt atcatggatt ctaaaacgga ttaccaggga tttcagtcga
     2281 tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat tttgtgccag
     2341 agtccttcga tagggacaag acaattgcac tgatcatgaa ctcctctgga tctactggtc
     2401 tgcctaaagg tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg catgccagag
     2461 atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt gttccattcc
     2521 atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt cgagtcgtct
     2581 taatgtatag atttgaagaa gagctgtttc tgaggagcct tcaggattac aagattcaaa
     2641 gtgcgctgct ggtgccaacc ctattctcct tcttcgccaa aagcactctg attgacaaat
     2701 acgatttatc taatttacac gaaattgctt ctggtggcgc tcccctctct aaggaagtcg
     2761 gggaagcggt tgccaagagg ttccatctgc caggtatcag gcaaggatat gggctcactg
     2821 agactacatc agctattctg attacacccg agggggatga taaaccgggc gcggtcggta
     2881 aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa acgctgggcg
     2941 ttaatcaaag aggcgaactg tgtgtgagag gtcctatgat tatgtccggt tatgtaaaca
     3001 atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct ggagacatag
     3061 cttactggga cgaagacgaa cacttcttca tcgttgaccg cctgaagtct ctgattaagt
     3121 acaaaggcta tcaggtggct cccgctgaat tggaatccat cttgctccaa caccccaaca
     3181 tcttcgacgc aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt cccgccgccg
     3241 ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat tacgtcgcca
     3301 gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac gaagtaccga
     3361 aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata aaggccaaga
     3421 agggcggaaa gatcgccgtg taagaattct gcagtcgacg gtaccgcggg cccgggatcc
     3481 accggatcta gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct
     3541 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     3601 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     3661 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     3721 tcttaacgcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     3781 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     3841 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     3901 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     3961 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     4021 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     4081 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     4141 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     4201 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     4261 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     4321 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     4381 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     4441 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4501 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4561 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4621 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     4681 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     4741 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     4801 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     4861 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     4921 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     4981 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     5041 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     5101 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     5161 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     5221 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     5281 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     5341 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     5401 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     5461 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     5521 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     5581 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     5641 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     5701 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     5761 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     5821 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     5881 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     5941 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     6001 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6061 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6121 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6181 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6241 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     6301 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6361 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6421 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6481 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6541 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6601 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6661 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6721 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     6781 cctgattctg tggataaccg tattaccgcc atgcattagt tattaatagt aatcaattac
     6841 ggggtcatta gttcatagcc catatatgga gttccg
//
