LOCUS       sequence-215843-        5839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally tagged 3x FLAG-REV7 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5839)
  AUTHORS   Noordermeer SM, Adam S, Setiaputra D, Barazas M, Pettitt SJ, Ling 
            AK, Olivieri M, Alvarez-Quilon A, Moatti N, Zimmermann M, Annunziato
            S, Krastev DB, Song F, Brandsma I, Frankum J, Brough R, Sherker A, 
            Landry S, Szilard RK, Munro MM, McEwan A, Goullet de Rugy T, Lin ZY,
            Hart T, Moffat J, Gingras AC, Martin A, van Attikum H, Jonkers J, 
            Lord CJ, Rottenberg S, Durocher D
  TITLE     The shieldin complex mediates 53BP1-dependent DNA repair.
  JOURNAL   Nature. 2018 Aug;560(7716):117-121. doi: 10.1038/s41586-018-0340-7. 
            Epub 2018 Jul 18.
  PUBMED    30022168
REFERENCE   2  (bases 1 to 5839)
  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: 
            "10.1038/s41586-018-0340-7"; journalName: "Nature"; date: "2018-08";
            volume: "560"; issue: "7716"; pages: "117-121"
COMMENT     Sequence Label: pDEST-FRT/TO-3xFLAG-REV7
FEATURES             Location/Qualifiers
     source          1..5839
                     /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"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1034..1057
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    1058..1082
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1725..1749)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1791..1808)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1797..2021
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2154..2173)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2305..2352
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2360..3382
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGDFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3512..3633
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3549..3568)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3603..3622
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3682..3698)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3682..3698)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3695..3717)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3706..3722
                     /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(3730..3736,3737..3754,3755..3760))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3775..3796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3913..3930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4084..4672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4164..4183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4843..5634,5635..5703))
                     /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     5466..5485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5704..5808)
                     /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 ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttccaccat ggactacaaa gaccatgacg gtgatacata
     1021 tgatcatgac atcgattaca aggatgacga tgacaagaca agtttgtaca aaaaagcagg
     1081 ctccaccatg accacgctca cacgacaaga cctcaacttt ggccaagtgg tggccgatgt
     1141 gctctgcgag ttcctggagg tggctgtgca tctcatcctc tacgtgcgcg aggtctaccc
     1201 cgtgggcatc ttccagaaac gcaagaagta caacgtgccg gtccagatgt cctgccaccc
     1261 ggagctgaat cagtatatcc aggacacgct gcactgcgtc aagccactcc tggagaagaa
     1321 tgatgtggag aaagtggtgg tggtgatttt ggataaagag caccgcccag tggagaaatt
     1381 cgtctttgag atcacccagc ctccactgct gtccatcagc tcagactcgc tgttgtctca
     1441 tgtggagcag ctgctccggg ccttcatcct gaagatcagc gtgtgcgatg ccgtcctgga
     1501 ccacaacccc ccaggctgta ccttcacagt cctggtgcac acgagagaag ccgccactcg
     1561 caacatggag aagatccagg tcatcaagga tttcccctgg atcctggcgg atgagcagga
     1621 tgtccacatg catgaccccc ggctgatacc actaaaaacc atgacgtcgg acattttaaa
     1681 gatgcagctt tacgtggaag agcgcgctca taaaggcagc taggacccag ctttcttgta
     1741 caaagtggtc tgactcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     1801 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     1861 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     1921 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     1981 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2041 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2101 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2161 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2221 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2281 ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag
     2341 tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     2401 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
     2461 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     2521 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     2581 ggaagtgctt gacattgggg atttcagcga gagcctgacc tattgcatct cccgccgtgc
     2641 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     2701 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     2761 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     2821 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     2881 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     2941 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     3001 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     3061 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     3121 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     3181 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     3241 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     3301 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     3361 tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta
     3421 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     3481 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     3541 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3601 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     3661 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     3721 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     3781 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     3841 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     3901 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     3961 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4021 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4081 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     4141 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     4201 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     4261 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     4321 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     4381 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     4441 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     4501 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     4561 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     4621 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     4681 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     4741 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     4801 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     4861 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     4921 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     4981 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5041 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     5101 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     5161 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     5221 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     5281 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     5341 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     5401 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     5461 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     5521 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     5581 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     5641 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     5701 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     5761 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     5821 aaaagtgcca cctgacgtc
//
