LOCUS       sequence-324308-        5546 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 5546)
  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 5546)
  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: ORF3a-EGFP
FEATURES             Location/Qualifiers
     source          1..5546
                     /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      231..240
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_feature    1062..1114
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      1122..1131
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1128..1130,1131..1133,1134..1847)
                     /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(1128..1130,1131..1133,1134..1844)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1173..1194)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1434..1453)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1781..1802
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1970..2091
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2007..2026)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2061..2080
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2098..2553)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2235..2256)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2447..2466
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2580..2684
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2686..3043
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2686..2877
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2709..2729)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2894..3029
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3078..3872
                     /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             3078..3872
                     /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(3132..3151)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3742..3761
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4060..4079)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4104..4151
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4480..5068
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4969..4988
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5243..5546
                     /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 gccaccatgg
      241 acctttttat gagaatcttt accatcggga ctgtcacact gaaacaggga gaaattaagg
      301 acgcgacccc gagtgacttt gtgagggcca ctgctacaat tcccatccaa gcctctcttc
      361 cattcggctg gctcatagtg ggtgtggcac tgctcgcggt attccagtca gcgtctaaga
      421 tcataacgct gaagaagaga tggcagttgg ctctcagcaa aggtgttcat ttcgtctgta
      481 acctgcttct tcttttcgtc actgtctatt ctcacttgct cttggttgca gccgggctgg
      541 aagccccgtt tctgtatctg tacgcgctcg tttacttcct ccagagcata aatttcgtac
      601 gaattattat gagactctgg ttgtgctgga agtgtcggag caagaatcct ctcttgtatg
      661 acgccaatta cttcctttgt tggcacacta attgttatga ttactgcata ccttacaata
      721 gcgttacctc atccatagta atcacctccg gtgacggaac aacgtcacca atctccgagc
      781 atgattacca gattggaggc tatacggaaa aatgggagag tggtgtcaag gactgtgtgg
      841 tgctgcatag ctatttcaca agcgattatt atcaactgta cagtacacag ttgtccacgg
      901 ataccggggt cgagcatgtc acatttttta tttataacaa gatcgttgat gagccagagg
      961 agcatgtgca gatccacacg atagatggaa gctccggtgt agtgaatcca gtcatggaac
     1021 cgatctatga cgagcccacc acgaccacat ctgtgcccct gctcgagctc aagcttcgaa
     1081 ttctgcagtc gacggtaccg cgggcccggg atccaccggt cgccaccatg gtgagcaagg
     1141 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     1201 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     1261 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     1321 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     1381 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     1441 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     1501 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     1561 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     1621 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     1681 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     1741 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     1801 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc ggccgcgact
     1861 ctagatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     1921 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     1981 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     2041 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa
     2101 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     2161 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     2221 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     2281 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     2341 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     2401 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     2461 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     2521 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     2581 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2641 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa
     2701 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     2761 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     2821 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     2881 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     2941 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     3001 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag
     3061 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     3121 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     3181 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     3241 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     3301 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     3361 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     3421 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     3481 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     3541 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     3601 aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     3661 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     3721 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     3781 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     3841 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     3901 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     3961 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4021 catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat
     4081 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg
     4141 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc
     4201 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag
     4261 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc
     4321 aggttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4381 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4441 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4501 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4561 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4621 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4681 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4741 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4801 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4861 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4921 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4981 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     5041 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     5101 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5161 taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg tcattagttc
     5221 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     5281 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     5341 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     5401 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
     5461 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
     5521 acgtattagt catcgctatt accatg
//
