LOCUS       sequence-352459-        4766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for cloning and expression of GFP-FLAG tagged proteins 
            (N-terminal tag). Confers resistance to G418..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4766)
  AUTHORS   Britton S, Coates J, Jackson SP
  TITLE     A new method for high-resolution imaging of Ku foci to decipher 
            mechanisms of DNA double-strand break repair.
  JOURNAL   J Cell Biol. 2013 Jul 29.
  PUBMED    23897892
REFERENCE   2  (bases 1 to 4766)
  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"; journalName: "J Cell 
            Biol. 2013 Jul 29."
COMMENT     Sequence Label: pEGFP-C1-FLAG
FEATURES             Location/Qualifiers
     source          1..4766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        98..289
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     246..266
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(291..395)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      422..877
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(509..528)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     719..740
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    884..1005
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(895..914)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     949..968
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1191..1214)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(join(1230..1940,1941..1943,1944..1946))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1230..1940,1941..1943,1944..1946))
                     /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(1272..1293)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1621..1640
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1880..1901
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1943..1952
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1991..2194)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2020..2040)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2195..2498
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(2673..3261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2753..2772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3590..3637
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3662..3681
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(3869..4663)
                     /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             complement(3869..4663)
                     /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(3980..3999)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4590..4609
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(4698..4766,1..289))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(4712..4766,1..81)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ctcggcctct gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact
       61 gggcggagtt aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt
      121 gagatgcatg ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt
      181 tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt
      241 tccacaccct aactgacaca cattccacag ctggttcttt ccgcctcagg actcttcctt
      301 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
      361 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
      421 gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
      481 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
      541 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
      601 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
      661 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
      721 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
      781 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
      841 aacgcgaatt ttaacaaaat attaacgctt acaatttacg cgttaagata cattgatgag
      901 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
      961 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     1021 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     1081 ctctacaaat gtggtatggc tgattatgat cagttatcta gatccggtgg atcccgggcc
     1141 cgcggtaccg tcgactgcag aattcgaagc tttccgcggc cgccctcgag cttatcgtcg
     1201 tcatccttgt aatcagatct gagtccggac ttgtacagct cgtccatgcc gagagtgatc
     1261 ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg
     1321 ctcagggcgg actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg
     1381 atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg
     1441 cggatcttga agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg
     1501 tggctgttgt agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg
     1561 atgcccttca gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg
     1621 gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc
     1681 atggcggact tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc
     1741 acgccgtagg tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg
     1801 cagatgaact tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg
     1861 ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg
     1921 aacagctcct cgcccttgct caccatggtg gcgaccggta gcgctagcgg atctgacggt
     1981 tcactaaacc agctctgctt atatagacct cccaccgtac acgcctaccg cccatttgcg
     2041 tcaatggggc ggagttgtta cgacattttg gaaagtcccg ttgattttgg tgccaaaaca
     2101 aactcccatt gacgtcaatg gggtggagac ttggaaatcc ccgtgagtca aaccgctatc
     2161 cacgcccatt gatgtactgc caaaaccgca tcaccatggt aatagcgatg actaatacgt
     2221 agatgtactg ccaagtagga aagtcccata aggtcatgta ctgggcataa tgccaggcgg
     2281 gccatttacc gtcattgacg tcaatagggg gcgtacttgg catatgatac acttgatgta
     2341 ctgccaagtg ggcagtttac cgtaaatact ccacccattg acgtcaatgg aaagtcccta
     2401 ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg
     2461 gtcagccagg cgggccattt accgtaagtt atgtaacgcg gaactccata tatgggctat
     2521 gaactaatga ccccgtaatt gattactatt aataactaat gcatggcggt aatacggtta
     2581 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2641 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2701 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2761 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2821 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2881 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2941 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3001 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3061 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3121 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3181 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3241 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3301 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3361 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaacct
     3421 gaggctatgg cagggcctgc cgccccgacg ttggctgcga gccctgggcc ttcacccgaa
     3481 cttggggggt ggggtgggga aaaggaagaa acgcgggcgt attggcccca atggggtctc
     3541 ggtggggtat cgacagagtg ccagccctgg gaccgaaccc cgcgtttatg aacaaacgac
     3601 ccaacaccgt gcgttttatt ctgtcttttt attgccgtca tagcgcgggt tccttccggt
     3661 attgtctcct tccgtgtttc agttagcctc cccctagggt gggcgaagaa ctccagcatg
     3721 agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc gaagcccaac
     3781 ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg
     3841 tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg cgatagaagg
     3901 cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt
     3961 cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga tagcggtccg
     4021 ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc accatgatat
     4081 tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc atgctcgcct
     4141 tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc agatcatcct
     4201 gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt
     4261 ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca tcagccatga
     4321 tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc ggcacttcgc
     4381 ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa
     4441 cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac
     4501 cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg
     4561 cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc ctctccaccc
     4621 aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac gatcctcatc
     4681 ctgtctcttg atcgatcttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc
     4741 tggaatagct cagaggccga ggcggc
//
