LOCUS       sequence-422356-        8008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human KU70-eGFP in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8008)
  AUTHORS   Shokrollahi M, Stanic M, Hundal A, Chan JNY, Urman D, Hakem A, 
            Garcia RE, Hao J, Maass PG, Dickson BC, Hande MP, Pujana MA, Hakem 
            R, Mekhail K
  TITLE     DNA double-strand break-capturing nuclear envelope tubules drive DNA
            repair
  JOURNAL   bioRxiv 2023.05.07.539750
REFERENCE   2  (bases 1 to 8008)
  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: "bioRxiv 
            2023.05.07.539750"
COMMENT     Sequence Label: KU70-eGFP
FEATURES             Location/Qualifiers
     source          1..8008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2791..2811
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2827..2829,2830..2832,2833..3543)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2827..2829,2830..2832,2833..3543)
                     /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(2872..2893)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3133..3152)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3480..3501
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3599..3616)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3605..3829
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3875..4303
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3962..3981)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4172..4193
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4312..4332)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4317..4646
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4497..4632
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4559..4578
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4713..5507
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4767..4786)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5377..5396
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5681..5802
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5718..5737)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5772..5791
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5851..5867)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5851..5867)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5864..5886)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5875..5891
                     /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(5899..5905,5906..5923,5924..5929))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5944..5965
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6082..6099)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6253..6841)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6333..6352)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7012..7803,7804..7872))
                     /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     7635..7654
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7873..7977)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccaccatgt cagggtggga gtcatattac aaaaccgagg gcgatgaaga agcagaggaa
      961 gaacaagaag agaaccttga agcaagtgga gactataaat attcaggaag agatagtttg
     1021 atttttttgg ttgatgcctc caaggctatg tttgaatctc agagtgaaga tgagttgaca
     1081 ccttttgaca tgagcatcca gtgtatccaa agtgtgtaca tcagtaagat cataagcagt
     1141 gatcgagatc tcttggctgt ggtgttctat ggtaccgaga aagacaaaaa ttcagtgaat
     1201 tttaaaaata tttacgtctt acaggagctg gataatccag gtgcaaaacg aattctagag
     1261 cttgaccagt ttaaggggca gcagggacaa aaacgtttcc aagacatgat gggccacgga
     1321 tctgactact cactcagtga agtgctgtgg gtctgtgcca acctctttag tgatgtccaa
     1381 ttcaagatga gtcataagag gatcatgctg ttcaccaatg aagacaaccc ccatggcaat
     1441 gacagtgcca aagccagccg ggccaggacc aaagccggtg atctccgaga tacaggcatc
     1501 ttccttgact tgatgcacct gaagaaacct gggggctttg acatatcctt gttctacaga
     1561 gatatcatca gcatagcaga ggatgaggac ctcagggttc actttgagga atccagcaag
     1621 ctagaagacc tgttgcggaa ggttcgcgcc aaggagacca ggaagcgagc actcagcagg
     1681 ttaaagctga agctcaacaa agatatagtg atctctgtgg gcatttataa tctggtccag
     1741 aaggctctca agcctcctcc aataaagctc tatcgggaaa caaatgaacc agtgaaaacc
     1801 aagacccgga cctttaatac aagtacaggc ggtttgcttc tgcctagcga taccaagagg
     1861 tctcagatct atgggagtcg tcagattata ctggagaaag aggaaacaga agagctaaaa
     1921 cggtttgatg atccaggttt gatgctcatg ggtttcaagc cgttggtact gctgaagaaa
     1981 caccattacc tgaggccctc cctgttcgtg tacccagagg agtcgctggt gattgggagc
     2041 tcaaccctgt tcagtgctct gctcatcaag tgtctggaga aggaggttgc agcattgtgc
     2101 agatacacac cccgcaggaa catccctcct tattttgtgg ctttggtgcc acaggaagaa
     2161 gagttggatg accagaaaat tcaggtgact cctccaggct tccagctggt ctttttaccc
     2221 tttgctgatg ataaaaggaa gatgcccttt actgaaaaaa tcatggcaac tccagagcag
     2281 gtgggcaaga tgaaggctat cgttgagaag cttcgcttca catacagaag tgacagcttt
     2341 gagaaccccg tgctgcagca gcacttcagg aacctggagg ccttggcctt ggatttgatg
     2401 gagccggaac aagcagtgga cctgacattg cccaaggttg aagcaatgaa taaaagactg
     2461 ggctccttgg tggatgagtt taaggagctt gtttacccac cagattacaa tcctgaaggg
     2521 aaagttacca agagaaaaca cgataatgaa ggttctggaa gcaaaaggcc caaggtggag
     2581 tattcagaag aggagctgaa gacccacatc agcaagggta cgctgggcaa gttcactgtg
     2641 cccatgctga aagaggcctg ccgggcttac gggctgaaga gtgggctgaa gaagcaggag
     2701 ctgctggaag ccctcaccaa gcacttccag gacatgctta agggaggtgg atctggaggt
     2761 ggatctggag gtggatctgg aggtggatct cccaagaaga agaggaaggt gggaggttct
     2821 accggtatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     2881 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     2941 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     3001 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     3061 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     3121 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     3181 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     3241 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     3301 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     3361 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     3421 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     3481 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     3541 aagggaggtt ctgttaactg actcgagtct agagggcccg tttaaacccg ctgatcagcc
     3601 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     3661 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     3721 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     3781 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     3841 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     3901 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     3961 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4021 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4081 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4141 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     4201 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     4261 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt
     4321 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4381 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     4441 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4501 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4561 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct
     4621 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat
     4681 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     4741 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     4801 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     4861 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc
     4921 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     4981 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     5041 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     5101 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     5161 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     5221 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg
     5281 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     5341 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     5401 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     5461 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     5521 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     5581 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     5641 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     5701 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     5761 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     5821 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     5881 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     5941 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6001 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6061 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     6121 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     6181 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     6241 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6301 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6361 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6421 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6481 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     6541 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     6601 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     6661 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     6721 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     6781 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     6841 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     6901 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     6961 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     7021 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     7081 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     7141 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     7201 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     7261 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7321 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7381 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7441 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7501 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     7561 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     7621 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     7681 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     7741 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     7801 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     7861 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     7921 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     7981 atttccccga aaagtgccac ctgacgtc
//
