LOCUS       sequence-215741-        7826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally tagged FLAG-SHLD2 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7826)
  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 7826)
  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-SHLD2
FEATURES             Location/Qualifiers
     source          1..7826
                     /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             993..1016
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3778..3795)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3784..4008
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4141..4160)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4292..4339
                     /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             4347..5369
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGDFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5499..5620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5536..5555)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5590..5609
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5669..5685)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5669..5685)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5682..5704)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5693..5709
                     /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(5717..5723,5724..5741,5742..5747))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5762..5783
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5900..5917)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6071..6659)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6151..6170)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6830..7621,7622..7690))
                     /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     7453..7472
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7691..7795)
                     /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 cttggtacca tggactacaa agacgatgac gataaagccg
     1021 ccagtttaaa cggcgcgcca agtggaggat ctcaagtcca cattttttgg ggtgctccaa
     1081 ttgctccact gaaaatcaca gtatcagaag acacagcttc tttaatgtct gttgctgacc
     1141 cctggaaaaa aattcagctt ttatacagtc aacattcttt atatctgaag gatgaaaaac
     1201 agcacaaaaa tcttgaaaac tataaagtcc cagaatctat tggttctcca gatcttagtg
     1261 gtcatttctt agcaaactgt atgaatagac atgttcatgt gaaagatgac tttgtacgtt
     1321 ctgtttctga aacacagaat atagaatccc agaagattca ctcctctaga ctgagtgata
     1381 taactagctc taatatgcaa atatgtggat ttaaaagcac agttccgcat ttcaccgaag
     1441 aagaaaagta tcaaaagctt ctcagtgaaa ataaaattag agatgaacag cctaaacatc
     1501 agccagatat atgtggtaag aactttaaca caaatttgtt tcagttgggc cataaatgtg
     1561 cagctgtgtt ggatttggtt tgtagtactg aaaaaattaa tatagggcct gaagtggtac
     1621 aaagagagtg tgtgccaaca gaatatcatg aaatacaaaa ccagtgtttg ggattatttt
     1681 cctcgaacgc agtagataag tcaaggtctg aagcagcagt taggaaggtc tcagacctta
     1741 aaatatcaac tgatacagaa tttctcagta taattacctc cagccaggtt gcttttttag
     1801 ctcaaaagaa agataaaagg cggagtcctg taaataaagg gaatgtaaac atggagactg
     1861 aaccaaaggc aagttacggg gagataagaa tacctgaaga gaattcgatt cagcttgatg
     1921 gttttacaga agcatatgaa agtggacaaa accaagcata ttcccttgaa ctttttagtc
     1981 ctgtttgtcc taaaacagaa aatagccgca ttcacataaa ctctgataaa ggtcttgaag
     2041 aacatacagg atctcaagaa cttttcagtt ctgaagatga actgccacca aatgagatac
     2101 gtattgagtt gtgtagctca ggaatactgt gttcccaact aaataccttc cacaaaagtg
     2161 ctattaaaag aagctgtacc tctgaagata aagtgggcca gtctgaagct ctatctagag
     2221 tccttcaagt agctaagaaa atgaagttga tttctaatgg aggagattct gctgtagaaa
     2281 tggatcggag aaatgtgtct gaatttaaga gtattaaaaa aacatcatta ataaaaaact
     2341 gtgattctaa aagccagaag tataattgtt tagtcatggt gctatctcca tgccatgtga
     2401 aggaaataaa cataaaattc ggaccaaatt ctggctctaa agtgccttta gcaacagtta
     2461 cagtaattga tcaatcagaa actaagaaga aggtttttct gtggaggact gcagcatttt
     2521 gggcatttac agtgtttctt ggagatataa ttttactcac agatgttgtt attcatgagg
     2581 accaatggat tggcgagaca gtactacaat caacatttag cagtcagtta ttaaatcttg
     2641 ggagttattc atctattcag cctgaagaat attccagtgt agttagtgaa gttgtacttc
     2701 aagacttact ggcatatgtg tcctcaaaac attcctacct cagagatctt cctccgaggc
     2761 agcctcagag ggtgaacagt atagactttg tagaattgga gcaccttcaa cctgatgtat
     2821 tagtccacgc agtactaaga gttgttgatt tcactatact gacagaggca gtatacagtt
     2881 atagaggaca gaagcagaaa aaagttatgt taacagtgga acaggcccaa gatcaacatt
     2941 atgcgcttgt attatggggt cctggagcag cctggtaccc tcaacttcaa aggaaaaaag
     3001 gttatatttg ggaatttaaa tatctttttg ttcagtgcaa ttacacacta gaaaacctag
     3061 aattgcatac aacgccttgg tcatcctgtg agtgcttgtt tgatgatgat ataagggcaa
     3121 ttacatttaa agcaaaattt caaaaaagtg caccctcctt tgtgaagata tcagacttag
     3181 caacccacct agaggataag tgttcaggag tggttctgat taaagcccag atttcagagc
     3241 tggcatttcc tattacagca tctcagaaga tagcgctaaa tgctcacagt tctctgaaga
     3301 gtattttttc ttctcttccc aacatcgtat atactggttg tgcaaaatgt ggattggaac
     3361 tagaaacaga tgagaacagg atctacaaac aatgttttag ctgcttgcca tttactatga
     3421 agaaaatata ttataggcca gcgttaatga ctgccattga tggaagacat gatgtttgta
     3481 tccgtgtaga atcaaagctg atagagaaga ttcttctcaa catttctgca gactgcctca
     3541 acagagtgat agttccttcc tcagagatca cctatgggat ggtcgtggca gacctgttcc
     3601 actccttgtt ggcagtcagc gcagaacctt gtgtattaaa gattcagagc ctttttgtgt
     3661 tagatgaaaa cagctatcca ttacaacaag atttctccct cctggatttt tatcctgaca
     3721 ttgtaaagca tggagccaat gcccgtctct gagggcccgt ttaaacccgc tgatcagcct
     3781 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     3841 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     3901 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     3961 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg
     4021 aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg
     4081 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     4141 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4201 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4261 aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt tcctattctc
     4321 tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg tctgtcgaga
     4381 agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg gagggcgaag
     4441 aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct
     4501 gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg gccgcgctcc
     4561 cgattccgga agtgcttgac attggggatt tcagcgagag cctgacctat tgcatctccc
     4621 gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc
     4681 agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag acgagcgggt
     4741 tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat ttcatatgcg
     4801 cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc gtcagtgcgt
     4861 ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc gaagtccggc
     4921 acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc cgcataacag
     4981 cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc gccaacatct
     5041 tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc gagcggaggc
     5101 atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc
     5161 aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg cagggtcgat
     5221 gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa
     5281 gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc
     5341 ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat tccaccgccg
     5401 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5461 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5521 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5581 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5641 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5701 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5761 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5821 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5881 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5941 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     6001 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6061 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6121 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6181 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6241 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6301 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6361 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6421 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6481 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6541 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6601 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6661 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6721 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6781 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6841 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6901 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6961 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     7021 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     7081 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7141 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7201 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7261 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7321 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7381 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7441 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7501 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7561 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7621 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7681 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7741 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7801 ttccccgaaa agtgccacct gacgtc
//
