LOCUS       sequence-393634-        4835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses eGFP fluorescent protein with a 3X flag tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4835)
  AUTHORS   Welden JR, Margvelani G, Arizaca Maquera KA, Gudlavalleti B, Miranda
            Sardon SC, Campos AR, Robil N, Lee DC, Hernandez AG, Wang WX, Di J, 
            de la Grange P, Nelson PT, Stamm S
  TITLE     RNA editing of microtubule-associated protein tau circular RNAs 
            promotes their translation and tau tangle formation.
  JOURNAL   Nucleic Acids Res. 2022 Dec 9;50(22):12979-12996. doi: 
            10.1093/nar/gkac1129.
  PUBMED    36533443
REFERENCE   2  (bases 1 to 4835)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2022-12-9- 9"; volume: "50"; issue: 
            "22"; pages: "12979-12996"
COMMENT     Sequence Label: pEGFP-C2_3X Flag
FEATURES             Location/Qualifiers
     source          1..4835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     promoter        613..631
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             647..712
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(713..715,716..718,719..1429)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(713..715,716..718,719..1429)
                     /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(758..779)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1019..1038)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1366..1387
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1443..1499
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    1623..1744
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1660..1679)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1714..1733
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1751..2206)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1888..1909)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2100..2119
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2233..2337
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2339..2696
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2339..2530
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2362..2382)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2547..2682
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2731..3525
                     /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             2731..3525
                     /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(2785..2804)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3395..3414
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3713..3732)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3757..3804
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4133..4721
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4622..4641
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca cctaatacga ctcactatag gaattccacc atggcggact acaaagacca
      661 tgacggtgat tataaagatc atgacatcga ttacaaggat gacgatgaca agatggtgag
      721 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
      781 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
      841 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
      901 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
      961 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     1021 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1081 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1141 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1201 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1261 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     1321 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     1381 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ccggccggac
     1441 tcagatctcg agctcaagct tcgaattctg cagtcgacgg taccgcgggc ccgggatcca
     1501 ccggatctag ataactgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     1561 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     1621 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     1681 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     1741 cttaacgcgt aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
     1801 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
     1861 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
     1921 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
     1981 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
     2041 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
     2101 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
     2161 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt
     2221 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     2281 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     2341 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     2401 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     2461 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2521 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     2581 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     2641 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc
     2701 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     2761 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     2821 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     2881 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     2941 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     3001 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     3061 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     3121 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     3181 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     3241 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     3301 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     3361 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3421 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3481 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     3541 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     3601 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     3661 agcgcgggga tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac
     3721 ggaaggagac aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa
     3781 cgcacggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg
     3841 tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac
     3901 cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc
     3961 ctgccatagc ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat
     4021 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4081 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4141 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4201 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4261 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4321 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4381 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4441 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4501 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4561 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4621 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     4681 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     4741 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     4801 ctgattctgt ggataaccgt attaccgcca tgcat
//
