LOCUS       sequence-325016-        5001 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 5001)
  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 5001)
  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: EGFP-Orf9b L52D
FEATURES             Location/Qualifiers
     source          1..5001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      243..252
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(249..251,252..254,255..965)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(249..251,252..254,255..965)
                     /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(294..315)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(555..574)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     902..923
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1425..1546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1462..1481)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1516..1535
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1553..2008)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1690..1711)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1902..1921
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2035..2139
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2141..2498
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2141..2332
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2164..2184)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2349..2484
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2533..3327
                     /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             2533..3327
                     /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(2587..2606)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3197..3216
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3515..3534)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3559..3606
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3935..4523
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4424..4443
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4698..5001
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
      301 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
      361 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
      421 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
      481 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
      541 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
      601 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
      661 tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc
      721 agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc
      781 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
      841 acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc
      901 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
      961 acaagtccgg actcagatct cgagctcaag cttcgaattc tatggacccc aaaatttctg
     1021 aaatgcaccc ggcgcttcgc ttggtggacc cccaaatcca actcgctgtc acgcggatgg
     1081 aaaacgcagt aggacgcgac caaaataatg tcggcccaaa agtgtatccg attatcctcc
     1141 gccttggcag tcccgacagc ctgaatatgg ccagaaagac tcttaacagc ctggaggaca
     1201 aagcgtttca gctgaccccc attgcggttc agatgactaa gctggcgaca actgaagagt
     1261 tgcccgacga atttgtggta gtaaccgtaa agtaaggatc caccggatct agataactga
     1321 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     1381 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     1441 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     1501 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta
     1561 agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac
     1621 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     1681 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     1741 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     1801 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     1861 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     1921 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     1981 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2041 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2101 ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga aagaaccagc
     2161 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     2221 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     2281 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     2341 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     2401 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
     2461 agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga gacaggatga
     2521 ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg
     2581 gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg
     2641 ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc
     2701 ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct
     2761 tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa
     2821 gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg
     2881 gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa
     2941 gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat
     3001 gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg
     3061 agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc
     3121 atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac
     3181 cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg
     3241 gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc
     3301 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
     3361 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
     3421 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
     3481 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
     3541 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
     3601 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     3661 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     3721 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
     3781 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3841 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3901 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3961 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4021 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4081 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4141 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4201 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4261 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4321 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4381 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4441 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4501 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4561 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4621 gtattaccgc catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc
     4681 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     4741 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     4801 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     4861 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     4921 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     4981 ttagtcatcg ctattaccat g
//
