LOCUS       sequence-325885-        4973 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 4973)
  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 4973)
  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: Nsp7-EGFP
FEATURES             Location/Qualifiers
     source          1..4973
                     /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"
     misc_feature    489..541
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      549..558
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(555..557,558..560,561..1274)
                     /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(555..557,558..560,561..1271)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(600..621)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(861..880)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1208..1229
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1397..1518
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1434..1453)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1488..1507
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1525..1980)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1662..1683)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1874..1893
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2007..2111
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2113..2470
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2113..2304
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2136..2156)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2321..2456
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2505..3299
                     /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             2505..3299
                     /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(2559..2578)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3169..3188
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3487..3506)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3531..3578
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3907..4495
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4396..4415
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4670..4973
                     /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 gccaccatga
      241 gcaagatgag cgacgtaaaa tgcacgtcag ttgttctctt gagcgtcctg caacaactca
      301 gagtggagtc tagttccaag ctctgggctc aatgcgttca gcttcacaat gacattctgt
      361 tggcgaaaga taccacggag gcattcgaga agatggtctc acttctgagc gttctcctgt
      421 caatgcaagg ggcggtcgat atcaataaat tgtgtgagga aatgctcgac aaccgggcga
      481 cattgcagct cgagctcaag cttcgaattc tgcagtcgac ggtaccgcgg gcccgggatc
      541 caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
      601 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
      661 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
      721 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
      781 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
      841 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
      901 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
      961 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     1021 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     1081 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     1141 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     1201 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     1261 agctgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga
     1321 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     1381 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     1441 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     1501 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     1561 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     1621 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     1681 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     1741 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     1801 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     1861 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     1921 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     1981 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     2041 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     2101 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     2161 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     2221 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     2281 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     2341 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2401 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2461 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     2521 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     2581 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     2641 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     2701 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     2761 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     2821 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     2881 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     2941 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     3001 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     3061 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     3121 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     3181 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     3241 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     3301 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     3361 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     3421 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     3481 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     3541 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     3601 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     3661 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     3721 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     3781 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     3841 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3901 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3961 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4021 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     4081 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4141 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4201 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4261 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4321 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4381 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4441 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4501 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4561 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcatt agttattaat
     4621 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     4681 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     4741 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     4801 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     4861 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     4921 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atg
//
