LOCUS       sequence-66853-e        7570 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For constitutive or doxycycline-inducible expression of mutant human
            Ku70 unable to bind DNA and resistant to siRNA. Confers resistance 
            to puro. Use T-REx cells for doxycycline-inducible expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7570)
  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 7570)
  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: pICE-EGFP-FLAG-Ku70siR-Mut6E
FEATURES             Location/Qualifiers
     source          1..7570
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      13..22
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(19..21,22..24,25..735)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(19..21,22..24,25..735)
                     /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(64..85)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(325..344)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     672..693
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             751..774
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2650..2667)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2656..2880
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2926..3354
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3013..3032)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3223..3244
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3363..3383)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3368..3697
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3548..3683
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3610..3629
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3749..4348
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3749..3768)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4245..4265
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4478..4599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4515..4534)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4569..4588
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4649..4665)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4649..4665)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4662..4684)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4673..4689
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4697..4703,4704..4721,4722..4727))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4742..4763
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4880..4897)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5051..5636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5131..5150)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5807..6598,6599..6667))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6430..6449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6668..6772)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6844..7223
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7224..7427
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7378..7398
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    7429..7447
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    7450..7468
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     7478..7502
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
ORIGIN
        1 agcttaccgg tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
       61 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      121 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      181 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
      241 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      301 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      361 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
      421 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
      481 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
      541 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
      601 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
      661 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
      721 gacgagctgt acaagtccgg actcagatct gattacaagg atgacgacga taagctcgag
      781 tcagggtggg agtcatatta caaaaccgag ggcgatgaag aagcagagga agaacaagaa
      841 gagaaccttg aagcaagtgg agactataaa tattcaggaa gagatagttt gatttttttg
      901 gttgatgcct ccaaggctat gtttgaatct caatccgagg acgaactcac cccttttgac
      961 atgagcatcc agtgtatcca aagtgtgtac atcagtaaga tcataagcag tgatcgagat
     1021 ctcttggctg tggtgttcta tggtaccgag aaagacaaaa attcagtgaa ttttaaaaat
     1081 atttacgtct tacaggagct ggataatcca ggtgcaaaac gaattctaga gcttgaccag
     1141 tttaaggggc agcagggaca aaaacgtttc caagacatga tgggccacgg atctgactac
     1201 tcactcagtg aagtgctgtg ggtctgtgcc aacctcttta gtgatgtcca attcaagatg
     1261 agtcataaga ggatcatgct gttcaccaat gaagacaacc cccatggcaa tgacagtgcc
     1321 aaagccagcc gggccaggac caaagccggt gatctccgag atacaggcat cttccttgac
     1381 ttgatgcacc tgaagaaacc tgggggcttt gacatatcct tgttctacag agatatcatc
     1441 agcatagcag aggatgagga cctcagggtt cactttgagg aatccagcaa gctagaagac
     1501 ctgttgcgga aggttcgcgc caaggagaca aggaagcgag cactcagcag gttaaagctg
     1561 aagctcaaca aagatatagt gatctctgtg ggcatttata atctggtcca gaaggctctc
     1621 gaacctcctc caatagagct ctatcgggaa acaaatgaac cagtgaaaac caaggagcgg
     1681 acctttaata caagtacagg cggtttgctt ctgcctagcg ataccaagag gtctcagatc
     1741 tatgggagtc gtcagattat actggaggaa gaggaaacag aagagctaga acggtttgat
     1801 gatccaggtt tgatgctcat gggtttcaag ccgttggtac tgctgaagaa acaccattac
     1861 ctgaggccct ccctgttcgt gtacccagag gagtcgctgg tgattgggag ctcaaccctg
     1921 ttcagtgctc tgctcatcaa gtgtctggag aaggaggttg cagcattgtg cagatacaca
     1981 cccgagagga acatccctcc ttattttgtg gctttggtgc cacaggaaga agagttggat
     2041 gaccagaaaa ttcaggtgac tcctccaggc ttccagctgg tctttttacc ctttgctgat
     2101 gataaaagga agatgccctt tactgaaaaa atcatggcaa ctccagagca ggtgggcaag
     2161 atgaaggcta tcgttgagaa gcttcgcttc acatacagaa gtgacagctt tgagaacccc
     2221 gtgctgcagc agcacttcag gaacctggag gccttggcct tggatttgat ggagccggaa
     2281 caagcagtgg acctgacatt gcccaaggtt gaagcaatga ataaaagact gggctccttg
     2341 gtggatgagt ttaaggagct tgtttaccca ccagattaca atcctgaagg gaaagttacc
     2401 aagagaaaac acgataatga aggttctgga agcaaaaggc ccaaggtgga gtattcagaa
     2461 gaggagctga agacccacat cagcaagggt acgctgggca agttcactgt gcccatgctg
     2521 aaagaggcct gccgggctta cgggctgaag agtgggctga agaagcagga gctgctggaa
     2581 gccctcacca agcacttcca ggactaagcg gccgcacgcg tctgcagtct agataaaccc
     2641 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     2701 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     2761 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     2821 gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg
     2881 cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg
     2941 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3001 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3061 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3121 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     3181 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3241 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     3301 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattaattct
     3361 gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3421 gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc
     3481 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac
     3541 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     3601 aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta
     3661 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc
     3721 cattttcgga tctgatcagc acgtgaccat gaccgagtac aagcccacgg tgcgcctcgc
     3781 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     3841 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     3901 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     3961 cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     4021 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     4081 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc
     4141 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     4201 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     4261 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     4321 gtgcatgacc cgcaagcccg gtgcctgaca cgtgctacga gatttcgatt ccaccgccgc
     4381 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     4441 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     4501 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4561 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tagccgtcga
     4621 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4681 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4741 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4801 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4861 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4921 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4981 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5041 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5101 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5161 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     5221 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     5281 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     5341 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     5401 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     5461 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     5521 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     5581 gtagcggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5641 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5701 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5761 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5821 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5881 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5941 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6001 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6061 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6121 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6181 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6241 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6301 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6361 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6421 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6481 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6541 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6601 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6661 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6721 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6781 cccgaaaagt gccacctgac gtcgacggat cgggagatcc tgtacgggcc agatatccgc
     6841 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
     6901 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
     6961 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
     7021 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
     7081 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
     7141 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
     7201 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
     7261 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
     7321 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
     7381 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
     7441 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
     7501 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
     7561 gcctccggca
//
