LOCUS       sequence-219959-        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"
     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(3544..3561)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3550..3774
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3907..3926)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4058..4105
                     /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             4113..5135
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5265..5386
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5302..5321)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5356..5375
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5435..5451)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5435..5451)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5448..5470)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5459..5475
                     /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(5483..5489,5490..5507,5508..5513))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5528..5549
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5666..5683)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5837..6425)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5917..5936)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6596..7387,7388..7456))
                     /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     7219..7238
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7457..7561)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7636..7655)
                     /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 gcgccaagtg gaggatctca agtccacatt ttttggggtg
      841 ctccaattgc tccactgaaa atcacagtat cagaagacac agcttcttta atgtctgttg
      901 ctgacccctg gaaaaaaatt cagcttttat acagtcaaca ttctttatat ctgaaggatg
      961 aaaaacagca caaaaatctt gaaaactata aagtcccaga atctattggt tctccagatc
     1021 ttagtggtca tttcttagca aactgtatga atagacatgt tcatgtgaaa gatgactttg
     1081 tacgttctgt ttctgaaaca cagaatatag aatcccagaa gattcactcc tctagactga
     1141 gtgatataac tagctctaat atgcaaatat gtggatttaa aagcacagtt ccgcatttca
     1201 ccgaagaaga aaagtatcaa aagcttctca gtgaaaataa aattagagat gaacagccta
     1261 aacatcagcc agatatatgt ggtaagaact ttaacacaaa tttgtttcag ttgggccata
     1321 aatgtgcagc tgtgttggat ttggtttgta gtactgaaaa aattaatata gggcctgaag
     1381 tggtacaaag agagtgtgtg ccaacagaat atcatgaaat acaaaaccag tgtttgggat
     1441 tattttcctc gaacgcagta gataagtcaa ggtctgaagc agcagttagg aaggtctcag
     1501 accttaaaat atcaactgat acagaatttc tcagtataat tacctccagc caggttgctt
     1561 ttttagctca aaagaaagat aaaaggcgga gtcctgtaaa taaagggaat gtaaacatgg
     1621 agactgaacc aaaggcaagt tacggggaga taagaatacc tgaagagaat tcgattcagc
     1681 ttgatggttt tacagaagca tatgaaagtg gacaaaacca agcatattcc cttgaacttt
     1741 ttagtcctgt ttgtcctaaa acagaaaata gccgcattca cataaactct gataaaggtc
     1801 ttgaagaaca tacaggatct caagaacttt tcagttctga agatgaactg ccaccaaatg
     1861 agatacgtat tgagttgtgt agctcaggaa tactgtgttc ccaactaaat accttccaca
     1921 aaagtgctat taaaagaagc tgtacctctg aagataaagt gggccagtct gaagctctat
     1981 ctagagtcct tcaagtagct aagaaaatga agttgatttc taatggagga gattctgctg
     2041 tagaaatgga tcggagaaat gtgtctgaat ttaagagtat taaaaaaaca tcattaataa
     2101 aaaactgtga ttctaaaagc cagaagtata attgtttagt catggtgcta tctccatgcc
     2161 atgtgaagga aataaacata aaattcggac caaattctgg ctctaaagtg cctttagcaa
     2221 cagttacagt aattgatcaa tcagaaacta agaagaaggt ttttctgtgg aggactgcag
     2281 cattttgggc atttacagtg tttcttggag atataatttt actcacagat gttgttattc
     2341 atgaggacca atggattggc gagacagtac tacaatcaac atttagcagt cagttattaa
     2401 atcttgggag ttattcatct attcagcctg aagaatattc cagtgtagtt agtgaagttg
     2461 tacttcaaga cttactggca tatgtgtcct caaaacattc ctacctcaga gatcttcctc
     2521 cgaggcagcc tcagagggtg aacagtatag actttgtaga attggagcac cttcaacctg
     2581 atgtattagt ccacgcagta ctaagagttg ttgatttcac tatactgaca gaggcagtat
     2641 acagttatag aggacagaag cagaaaaaag ttatgttaac agtggaacag gcccaagatc
     2701 aacattatgc gcttgtatta tggggtcctg gagcagcctg gtaccctcaa cttcaaagga
     2761 aaaaaggtta tatttgggaa tttaaatatc tttttgttca gtgcaattac acactagaaa
     2821 acctagaatt gcatacaacg ccttggtcat cctgtgagtg cttgtttgat gatgatataa
     2881 gggcaattac atttaaagca aaatttcaaa aaagtgcacc ctcctttgtg aagatatcag
     2941 acttagcaac ccacctagag gataagtgtt caggagtggt tctgattaaa gcccagattt
     3001 cagagctggc atttcctatt acagcatctc agaagatagc gctaaatgct cacagttctc
     3061 tgaagagtat tttttcttct cttcccaaca tcgtatatac tggttgtgca aaatgtggat
     3121 tggaactaga aacagatgag aacaggatct acaaacaatg ttttagctgc ttgccattta
     3181 ctatgaagaa aatatattat aggccagcgt taatgactgc cattgatgga agacatgatg
     3241 tttgtatccg tgtagaatca aagctgatag agaagattct tctcaacatt tctgcagact
     3301 gcctcaacag agtgatagtt ccttcctcag agatcaccta tgggatggtc gtggcagacc
     3361 tgttccactc cttgttggca gtcagcgcag aaccttgtgt attaaagatt cagagccttt
     3421 ttgtgttaga tgaaaacagc tatccattac aacaagattt ctccctcctg gatttttatc
     3481 ctgacattgt aaagcatgga gccaatgccc gtctctgagg gcccgtttaa acccgctgat
     3541 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3601 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3661 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3721 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3781 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3841 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3901 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3961 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4021 ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct
     4081 attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg
     4141 tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg
     4201 gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa
     4261 atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg
     4321 cgctcccgat tccggaagtg cttgacattg gggagttcag cgagagcctg acctattgca
     4381 tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg
     4441 ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga
     4501 gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca
     4561 tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca
     4621 gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag
     4681 tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca
     4741 taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca
     4801 acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc
     4861 ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc
     4921 ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg
     4981 gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc
     5041 gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc
     5101 gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca
     5161 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5221 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     5281 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5341 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5401 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5461 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5521 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5581 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5641 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5701 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5761 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5821 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5881 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5941 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6001 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6061 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6121 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6181 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6241 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6301 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6361 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6421 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6481 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6541 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6601 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6661 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6721 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6781 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6841 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6901 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6961 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7021 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7081 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7141 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7201 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7261 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7321 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7381 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7441 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7501 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7561 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct
     7621 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc
     7681 tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca
     7741 aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg cgctgcttcg
     7801 cgatgtacgg gccagatata cgcgtt
//
