LOCUS       sequence-218643-        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"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             800..823
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    824..848
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1491..1515)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1557..1574)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1563..1787
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1920..1939)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2071..2118
                     /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             2126..3148
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3278..3399
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3315..3334)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3369..3388
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3448..3464)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3448..3464)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3461..3483)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3472..3488
                     /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(3496..3502,3503..3520,3521..3526))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3541..3562
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3679..3696)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3850..4438)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3930..3949)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4609..5400,5401..5469))
                     /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     5232..5251
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5470..5574)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5649..5668)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagcttc caccatggac tacaaagacc atgacggtga
      781 tacatatgat catgacatcg attacaagga tgacgatgac aagacaagtt tgtacaaaaa
      841 agcaggctcc accatgacca cgctcacacg acaagacctc aactttggcc aagtggtggc
      901 cgatgtgctc tgcgagttcc tggaggtggc tgtgcatctc atcctctacg tgcgcgaggt
      961 ctaccccgtg ggcatcttcc agaaacgcaa gaagtacaac gtgccggtcc agatgtcctg
     1021 ccacccggag ctgaatcagt atatccagga cacgctgcac tgcgtcaagc cactcctgga
     1081 gaagaatgat gtggagaaag tggtggtggt gattttggat aaagagcacc gcccagtgga
     1141 gaaattcgtc tttgagatca cccagcctcc actgctgtcc atcagctcag actcgctgtt
     1201 gtctcatgtg gagcagctgc tccgggcctt catcctgaag atcagcgtgt gcgatgccgt
     1261 cctggaccac aaccccccag gctgtacctt cacagtcctg gtgcacacga gagaagccgc
     1321 cactcgcaac atggagaaga tccaggtcat caaggatttc ccctggatcc tggcggatga
     1381 gcaggatgtc cacatgcatg acccccggct gataccacta aaaaccatga cgtcggacat
     1441 tttaaagatg cagctttacg tggaagagcg cgctcataaa ggcagctagg acccagcttt
     1501 cttgtacaaa gtggtctgac tcgagtctag agggcccgtt taaacccgct gatcagcctc
     1561 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     1621 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     1681 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     1741 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     1801 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     1861 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1921 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1981 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     2041 acttgattag ggtgatggtt cacgtaccta gaagttccta ttccgaagtt cctattctct
     2101 agaaagtata ggaacttcct tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa
     2161 gtttctgatc gaaaagttcg acagcgtctc cgacctgatg cagctctcgg agggcgaaga
     2221 atctcgtgct ttcagcttcg atgtaggagg gcgtggatat gtcctgcggg taaatagctg
     2281 cgccgatggt ttctacaaag atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc
     2341 gattccggaa gtgcttgaca ttggggagtt cagcgagagc ctgacctatt gcatctcccg
     2401 ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc gaactgcccg ctgttctgca
     2461 gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat cttagccaga cgagcgggtt
     2521 cggcccattc ggaccgcaag gaatcggtca atacactaca tggcgtgatt tcatatgcgc
     2581 gattgctgat ccccatgtgt atcactggca aactgtgatg gacgacaccg tcagtgcgtc
     2641 cgtcgcgcag gctctcgatg agctgatgct ttgggccgag gactgccccg aagtccggca
     2701 cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg gacaatggcc gcataacagc
     2761 ggtcattgac tggagcgagg cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt
     2821 cttctggagg ccgtggttgg cttgtatgga gcagcagacg cgctacttcg agcggaggca
     2881 tccggagctt gcaggatcgc cgcggctccg ggcgtatatg ctccgcattg gtcttgacca
     2941 actctatcag agcttggttg acggcaattt cgatgatgca gcttgggcgc agggtcgatg
     3001 cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag
     3061 cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc
     3121 cagcactcgt ccgagggcaa aggaatagca cgtactacga gatttcgatt ccaccgccgc
     3181 cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca
     3241 gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa
     3301 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     3361 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac
     3421 ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     3481 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     3541 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     3601 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     3661 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     3721 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     3781 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     3841 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     3901 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     3961 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     4021 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     4081 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     4141 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     4201 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     4261 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     4321 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     4381 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     4441 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     4501 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     4561 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     4621 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     4681 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     4741 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     4801 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     4861 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     4921 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     4981 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     5041 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     5101 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     5161 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     5221 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     5281 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     5341 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     5401 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     5461 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     5521 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     5581 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     5641 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     5701 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     5761 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     5821 cgggccagat atacgcgtt
//
