LOCUS       sequence-325883-        5318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression and localization.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5318)
  AUTHORS   Miserey-Lenkei S, Trajkovic K, D'Ambrosio JM, Patel AJ, Copic A, 
            Mathur P, Schauer K, Goud B, Albanese V, Gautier R, Subra M, Kovacs 
            D, Barelli H, Antonny B
  TITLE     A comprehensive library of fluorescent constructs of SARS-CoV-2 
            proteins and their initial characterisation in different cell types.
  JOURNAL   Biol Cell. 2021 Jul;113(7):311-328. doi: 10.1111/boc.202000158. Epub
            2021 May 10.
  PUBMED    33666950
REFERENCE   2  (bases 1 to 5318)
  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: 
            "10.1111/boc.202000158"; journalName: "Biol Cell"; date: "2021-07"; 
            volume: "113"; issue: "7"; pages: "311-328"
COMMENT     Sequence Label: Nsp8-EGFP
FEATURES             Location/Qualifiers
     source          1..5318
                     /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"
     regulatory      535..544
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1138..1190
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1198..1207
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1204..1206,1207..1209,1210..1923)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1204..1206,1207..1209,1210..1920)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1249..1270)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1510..1529)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1857..1878
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2046..2167
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2083..2102)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2137..2156
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2174..2629)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2311..2332)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2523..2542
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2656..2760
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2762..3119
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2762..2953
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2785..2805)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2970..3105
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3154..3948
                     /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             3154..3948
                     /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(3208..3227)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3818..3837
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4136..4155)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4180..4227
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4556..5144
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5045..5064
                     /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 gcagagctgg tttagtgaac cgtcagatcc gctagccacc
      541 atggcaatcg catctgaatt ttctagtctc ccctcctatg cagcgtttgc aactgcccag
      601 gaggcatacg agcaggctgt tgcgaacggc gactccgaag tggtgctgaa gaagctgaaa
      661 aagtctctga acgtcgcaaa atccgagttt gatagagatg cagcaatgca aagaaagctg
      721 gagaaaatgg cagatcaagc tatgacacaa atgtacaagc aggcccgctc agaggataaa
      781 cgagctaaag ttacctccgc aatgcaaact atgctgttca ccatgctgag aaagctggac
      841 aacgacgcgc tgaacaatat aattaataat gcgcgggacg ggtgcgtacc cttgaatatt
      901 attcccctta cgactgccgc gaagctcatg gttgtcatac cagattataa cacttacaag
      961 aatacgtgtg atggtacgac gttcacttat gcgtctgcat tgtgggaaat tcagcaagtg
     1021 gtagacgctg attccaagat agtacagttg tccgagatta gcatggacaa cagtcccaat
     1081 ctcgcttggc ctctgatcgt taccgcattg agggctaaca gcgcagtcaa actgcagctc
     1141 gagctcaagc ttcgaattct gcagtcgacg gtaccgcggg cccgggatcc accggtcgcc
     1201 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1261 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1321 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1381 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1441 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1501 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1561 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1621 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     1681 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     1741 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1801 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     1861 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     1921 taaagcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     1981 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     2041 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     2101 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     2161 tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     2221 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2281 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2341 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2401 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2461 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2521 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2581 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     2641 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2701 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2761 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     2821 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     2881 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     2941 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3001 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     3061 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     3121 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3181 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3241 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3301 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     3361 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3421 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3481 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3541 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3601 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3661 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     3721 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3781 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     3841 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     3901 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     3961 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4021 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4081 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     4141 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     4201 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     4261 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     4321 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     4381 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     4441 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4501 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4561 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4621 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4681 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4741 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4801 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4861 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4921 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4981 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5041 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5101 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5161 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5221 cccctgattc tgtggataac cgtattaccg ccatgcatta gttattaata gtaatcaatt
     5281 acggggtcat tagttcatag cccatatatg gagttccg
//
