LOCUS       sequence-197817-        6543 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid for cloning and epression of GFP-FLAG tagged human Ku70 
            (N-terminal tag). Confers resistance to G418..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6543)
  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 6543)
  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-Ku70
FEATURES             Location/Qualifiers
     source          1..6543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        64..367
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        368..571
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     522..542
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      610..619
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(616..618,619..621,622..1332)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(616..618,619..621,622..1332)
                     /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(661..682)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(922..941)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1269..1290
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1348..1371
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3334..3455
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3371..3390)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3425..3444
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3462..3917)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3599..3620)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3811..3830
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3944..4048
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4050..4407
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4050..4241
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4073..4093)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4258..4393
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4442..5236
                     /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             4442..5236
                     /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(4496..4515)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5106..5125
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5424..5443)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5468..5515
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5844..6432
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6333..6352
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cattagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
       61 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
      121 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
      181 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
      241 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
      301 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
      361 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
      421 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
      481 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
      541 tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg
      601 ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      661 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
      721 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
      781 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
      841 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
      901 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
      961 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1021 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     1081 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     1141 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1201 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     1261 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1321 gagctgtaca agtccggact cagatctgat tacaaggatg acgacgataa gctcgagtca
     1381 gggtgggagt catattacaa aaccgagggc gatgaagaag cagaggaaga acaagaagag
     1441 aaccttgaag caagtggaga ctataaatat tcaggaagag atagtttgat ttttttggtt
     1501 gatgcctcca aggctatgtt tgaatctcag agtgaagatg agttgacacc ttttgacatg
     1561 agcatccagt gtatccaaag tgtgtacatc agtaagatca taagcagtga tcgagatctc
     1621 ttggctgtgg tgttctatgg taccgagaaa gacaaaaatt cagtgaattt taaaaatatt
     1681 tacgtcttac aggagctgga taatccaggt gcaaaacgaa ttctagagct tgaccagttt
     1741 aaggggcagc agggacaaaa acgtttccaa gacatgatgg gccacggatc tgactactca
     1801 ctcagtgaag tgctgtgggt ctgtgccaac ctctttagtg atgtccaatt caagatgagt
     1861 cataagagga tcatgctgtt caccaatgaa gacaaccccc atggcaatga cagtgccaaa
     1921 gccagccggg ccaggaccaa agccggtgat ctccgagata caggcatctt ccttgacttg
     1981 atgcacctga agaaacctgg gggctttgac atatccttgt tctacagaga tatcatcagc
     2041 atagcagagg atgaggacct cagggttcac tttgaggaat ccagcaagct agaagacctg
     2101 ttgcggaagg ttcgcgccaa ggagacaagg aagcgagcac tcagcaggtt aaagctgaag
     2161 ctcaacaaag atatagtgat ctctgtgggc atttataatc tggtccagaa ggctctcaag
     2221 cctcctccaa taaagctcta tcgggaaaca aatgaaccag tgaaaaccaa gacccggacc
     2281 tttaatacaa gtacaggcgg tttgcttctg cctagcgata ccaagaggtc tcagatctat
     2341 gggagtcgtc agattatact ggagaaagag gaaacagaag agctaaaacg gtttgatgat
     2401 ccaggtttga tgctcatggg tttcaagccg ttggtactgc tgaagaaaca ccattacctg
     2461 aggccctccc tgttcgtgta cccagaggag tcgctggtga ttgggagctc aaccctgttc
     2521 agtgctctgc tcatcaagtg tctggagaag gaggttgcag cattgtgcag atacacaccc
     2581 cgcaggaaca tccctcctta ttttgtggct ttggtgccac aggaagaaga gttggatgac
     2641 cagaaaattc aggtgactcc tccaggcttc cagctggtct ttttaccctt tgctgatgat
     2701 aaaaggaaga tgccctttac tgaaaaaatc atggcaactc cagagcaggt gggcaagatg
     2761 aaggctatcg ttgagaagct tcgcttcaca tacagaagtg acagctttga gaaccccgtg
     2821 ctgcagcagc acttcaggaa cctggaggcc ttggccttgg atttgatgga gccggaacaa
     2881 gcagtggacc tgacattgcc caaggttgaa gcaatgaata aaagactggg ctccttggtg
     2941 gatgagttta aggagcttgt ttacccacca gattacaatc ctgaagggaa agttaccaag
     3001 agaaaacacg ataatgaagg ttctggaagc aaaaggccca aggtggagta ttcagaagag
     3061 gagctgaaga cccacatcag caagggtacg ctgggcaagt tcactgtgcc catgctgaaa
     3121 gaggcctgcc gggcttacgg gctgaagagt gggctgaaga agcaggagct gctggaagcc
     3181 ctcaccaagc acttccagga ctaaggatcc accggatcta gataactgat cataatcagc
     3241 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     3301 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     3361 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     3421 agttgtggtt tgtccaaact catcaatgta tcttaacgcg taaattgtaa gcgttaatat
     3481 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     3541 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     3601 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     3661 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     3721 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     3781 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     3841 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     3901 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3961 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4021 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     4081 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4141 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     4201 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4261 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4321 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     4381 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     4441 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     4501 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     4561 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     4621 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     4681 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     4741 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     4801 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     4861 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     4921 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     4981 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     5041 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     5101 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     5161 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     5221 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     5281 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     5341 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     5401 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     5461 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     5521 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     5581 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     5641 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     5701 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5761 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5821 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5881 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5941 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     6001 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     6061 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     6121 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6181 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6241 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6301 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6361 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6421 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6481 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6541 atg
//
