LOCUS       sequence-218638-        5876 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally tagged FLAG-SHLD3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5876)
  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 5876)
  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: pcDNA5-FRT/TO-FLAG-SHLD3
FEATURES             Location/Qualifiers
     source          1..5876
                     /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             759..782
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1594..1611)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1600..1824
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1957..1976)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2108..2155
                     /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             2163..3185
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3315..3436
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3352..3371)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3406..3425
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3485..3501)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3485..3501)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3498..3520)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3509..3525
                     /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(3533..3539,3540..3557,3558..3563))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3578..3599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3716..3733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3887..4475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3967..3986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4646..5437,5438..5506))
                     /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     5269..5288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5507..5611)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5686..5705)
                     /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 cttaagcttg gtaccatgga ctacaaagac gatgacgata
      781 aagccgccag tttaaacggc gcgccaacta cagaagtaat attacattat cgaccatgtg
      841 agagtgatcc cacacaactg ccaaaaattg cagaaaaagc aattcaagac tttcctactc
      901 gtccgctatc aagatttata ccttggtttc catatgatgg gtccaagctt ccactcagac
      961 ctaaaagatc accacctgtg atttctgaag aggcagctga agatgtgaaa cagtacttaa
     1021 ccatttcaga acatgatgct aagtcacaca gttatgattg cacagtagat ctattggagt
     1081 ttcaacctag cttgaaaaag cagcatttaa cctggtcaca cacactgaag gaacagacta
     1141 attctggaaa tctgggtaaa caatcagaaa agggaaaaca gcacaagagg agatcttgga
     1201 gtatttccct tcccagcaat aattgtacta aaaacgtttc tcctttgtct aaaaaattgc
     1261 aagatagttt aaaggcacta aatttacact cactttatag agcaagatgg acaatagaac
     1321 acactatttg taacagccaa actctggaag acatttggac aaaactcaat caaattatta
     1381 ggcacaatga acttccatct tgtaatgcta caattcagag gcatttaggc caaatatggg
     1441 tgttctgtga tattatgtat tgtgaatatg tgggaagtct tcttaaagga agattagctc
     1501 ttactggaaa aattaattta tttgtgcata aatatggtgt tatttttagt atgtagctcg
     1561 agtctagagg gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     1621 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     1681 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     1741 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     1801 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     1861 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     1921 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     1981 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     2041 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     2101 gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg
     2161 ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
     2221 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
     2281 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
     2341 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
     2401 gggagttcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     2461 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
     2521 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     2581 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     2641 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     2701 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     2761 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     2821 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     2881 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     2941 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     3001 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     3061 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     3121 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     3181 aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     3241 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     3301 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     3361 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     3421 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     3481 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     3541 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     3601 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3661 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3721 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3781 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3841 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3901 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3961 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4021 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4081 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4141 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4201 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4261 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4321 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4381 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4441 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4501 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4561 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4621 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4681 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4741 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4801 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4861 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4921 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4981 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5041 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5101 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5161 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5221 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5281 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5341 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5401 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5461 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5521 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5581 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     5641 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     5701 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     5761 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     5821 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgtt
//
