LOCUS       sequence-215743-        8177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses N-terminally tagged Streptag-HA-CBP-SHLD2 in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8177)
  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 8177)
  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: pGLUE-SHLD2
FEATURES             Location/Qualifiers
     source          1..8177
                     /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..612
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        613..816
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     767..787
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     861..880
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        861..879
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      921..930
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             927..1040
                     /codon_start=1
                     /product="streptavidin-binding peptide"
                     /label=SBP
                     /note="selected from a peptide library; binds streptavidin 
                     with nanomolar affinity (Keefe et al., 2001)"
                     /translation="MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP"
     CDS             1059..1079
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1086..1112
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             1086..1112
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1113..1190
                     /codon_start=1
                     /product="calmodulin-binding peptide"
                     /label=CBP
                     /note="derived from skeletal muscle myosin light chain 
                     kinase; binds calmodulin with nanomolar affinity in the 
                     presence of calcium"
                     /translation="KRRWKKNFIAVSAANRFKKISSSGAL"
     intron          4036..4265
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     4248..4267
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(4323..4897,4898..4900,4901..4909)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4490..4507)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4717..4736
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4929..5528
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     primer_bind     complement(4929..4948)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5425..5445
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5664..5681)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5670..5894
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5941..5960
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6020..6036)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6020..6036)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6033..6055)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6044..6060
                     /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(6068..6074,6075..6092,6093..6098))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6113..6134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6251..6268)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6422..7010)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6502..6521)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7181..7972,7973..8041))
                     /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     7804..7823
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8042..8146)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct 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 tagtatcaat tacggggtca ttagttcata gcccatatat
      301 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccacgacccc
      361 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      421 tgacgtcaat gggtggacta tttacggtaa actgcccact tggcagtaca tcaagtgtat
      481 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
      541 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
      601 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
      661 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
      721 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
      781 aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag agaacccact
      841 gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc ttggtaccga
      901 gctcggatcg atttaattaa gccaccatgg acgagaagac caccggctgg cggggcggcc
      961 acgtggtgga gggcctggcc ggcgagctgg agcagctgcg ggcccggctg gagcaccacc
     1021 cccagggcca gcgggagccc ggcggaagcc ccggtggcga gaacctgtac ttccagggcg
     1081 caagctaccc ctacgacgtg cccgactacg ccaagcggcg gtggaagaag aacttcatcg
     1141 ccgtgagcgc cgccaaccgg ttcaagaaga tcagcagcag cggcgccctg attgatgggg
     1201 gatctggccc cggcggcggc gcgccaagtg gaggatctca agtccacatt ttttggggtg
     1261 ctccaattgc tccactgaaa atcacagtat cagaagacac agcttcttta atgtctgttg
     1321 ctgacccctg gaaaaaaatt cagcttttat acagtcaaca ttctttatat ctgaaggatg
     1381 aaaaacagca caaaaatctt gaaaactata aagtcccaga atctattggt tctccagatc
     1441 ttagtggtca tttcttagca aactgtatga atagacatgt tcatgtgaaa gatgactttg
     1501 tacgttctgt ttctgaaaca cagaatatag aatcccagaa gattcactcc tctagactga
     1561 gtgatataac tagctctaat atgcaaatat gtggatttaa aagcacagtt ccgcatttca
     1621 ccgaagaaga aaagtatcaa aagcttctca gtgaaaataa aattagagat gaacagccta
     1681 aacatcagcc agatatatgt ggtaagaact ttaacacaaa tttgtttcag ttgggccata
     1741 aatgtgcagc tgtgttggat ttggtttgta gtactgaaaa aattaatata gggcctgaag
     1801 tggtacaaag agagtgtgtg ccaacagaat atcatgaaat acaaaaccag tgtttgggat
     1861 tattttcctc gaacgcagta gataagtcaa ggtctgaagc agcagttagg aaggtctcag
     1921 accttaaaat atcaactgat acagaatttc tcagtataat tacctccagc caggttgctt
     1981 ttttagctca aaagaaagat aaaaggcgga gtcctgtaaa taaagggaat gtaaacatgg
     2041 agactgaacc aaaggcaagt tacggggaga taagaatacc tgaagagaat tcgattcagc
     2101 ttgatggttt tacagaagca tatgaaagtg gacaaaacca agcatattcc cttgaacttt
     2161 ttagtcctgt ttgtcctaaa acagaaaata gccgcattca cataaactct gataaaggtc
     2221 ttgaagaaca tacaggatct caagaacttt tcagttctga agatgaactg ccaccaaatg
     2281 agatacgtat tgagttgtgt agctcaggaa tactgtgttc ccaactaaat accttccaca
     2341 aaagtgctat taaaagaagc tgtacctctg aagataaagt gggccagtct gaagctctat
     2401 ctagagtcct tcaagtagct aagaaaatga agttgatttc taatggagga gattctgctg
     2461 tagaaatgga tcggagaaat gtgtctgaat ttaagagtat taaaaaaaca tcattaataa
     2521 aaaactgtga ttctaaaagc cagaagtata attgtttagt catggtgcta tctccatgcc
     2581 atgtgaagga aataaacata aaattcggac caaattctgg ctctaaagtg cctttagcaa
     2641 cagttacagt aattgatcaa tcagaaacta agaagaaggt ttttctgtgg aggactgcag
     2701 cattttgggc atttacagtg tttcttggag atataatttt actcacagat gttgttattc
     2761 atgaggacca atggattggc gagacagtac tacaatcaac atttagcagt cagttattaa
     2821 atcttgggag ttattcatct attcagcctg aagaatattc cagtgtagtt agtgaagttg
     2881 tacttcaaga cttactggca tatgtgtcct caaaacattc ctacctcaga gatcttcctc
     2941 cgaggcagcc tcagagggtg aacagtatag actttgtaga attggagcac cttcaacctg
     3001 atgtattagt ccacgcagta ctaagagttg ttgatttcac tatactgaca gaggcagtat
     3061 acagttatag aggacagaag cagaaaaaag ttatgttaac agtggaacag gcccaagatc
     3121 aacattatgc gcttgtatta tggggtcctg gagcagcctg gtaccctcaa cttcaaagga
     3181 aaaaaggtta tatttgggaa tttaaatatc tttttgttca gtgcaattac acactagaaa
     3241 acctagaatt gcatacaacg ccttggtcat cctgtgagtg cttgtttgat gatgatataa
     3301 gggcaattac atttaaagca aaatttcaaa aaagtgcacc ctcctttgtg aagatatcag
     3361 acttagcaac ccacctagag gataagtgtt caggagtggt tctgattaaa gcccagattt
     3421 cagagctggc atttcctatt acagcatctc agaagatagc gctaaatgct cacagttctc
     3481 tgaagagtat tttttcttct cttcccaaca tcgtatatac tggttgtgca aaatgtggat
     3541 tggaactaga aacagatgag aacaggatct acaaacaatg ttttagctgc ttgccattta
     3601 ctatgaagaa aatatattat aggccagcgt taatgactgc cattgatgga agacatgatg
     3661 tttgtatccg tgtagaatca aagctgatag agaagattct tctcaacatt tctgcagact
     3721 gcctcaacag agtgatagtt ccttcctcag agatcaccta tgggatggtc gtggcagacc
     3781 tgttccactc cttgttggca gtcagcgcag aaccttgtgt attaaagatt cagagccttt
     3841 ttgtgttaga tgaaaacagc tatccattac aacaagattt ctccctcctg gatttttatc
     3901 ctgacattgt aaagcatgga gccaatgccc gtctctgagc ggccgcgtcg acggaattcg
     3961 cccttttcgg atccactagt aacggccgcc agtgtgctgg aattaattcg ctgtctgcga
     4021 gggccagctg ttggggtgag tactccctct caaaagcggg catgacttct gcgctaagat
     4081 tgtcagtttc caaaaacgag gaggatttga tattcacctg gcccgcggtg atgcctttga
     4141 gggtggccgc gtccatctgg tcagaaaaga caatcttttt gttgtcaagc ttgaggtgtg
     4201 gcaggcttga gatctggcca tacacttgag tgacaatgac atccactttg cctttctctc
     4261 cacaggtgtc cactcccagg tccaactgca ggtcgagcat gcatctaggg cggccaattc
     4321 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg
     4381 tgtgcgtttg tctatatgtg attttccacc atattgccgt cttttggcaa tgtgagggcc
     4441 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa
     4501 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
     4561 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
     4621 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
     4681 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
     4741 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
     4801 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
     4861 ggggacgtgg ttttcctttg aaaaacacga tgataagctt gccacaaccc acaaggagac
     4921 gaccttccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
     4981 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     5041 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     5101 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     5161 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     5221 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     5281 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     5341 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     5401 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     5461 tcaccgccga cgtcgagtgc ccgaaggacc gcgcgacctg gtgcatgacc cgcaagcccg
     5521 gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg
     5581 gctccgaccg aagccgaccc gggcggcccc gccgaccccg cacccgcccc cgaggcccac
     5641 cgactctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     5701 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     5761 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     5821 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     5881 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgagtg cattctagtt
     5941 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     6001 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     6061 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     6121 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6181 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     6241 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     6301 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     6361 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     6421 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     6481 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     6541 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     6601 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     6661 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     6721 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     6781 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     6841 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     6901 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     6961 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     7021 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     7081 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     7141 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     7201 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     7261 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     7321 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     7381 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     7441 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     7501 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     7561 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     7621 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     7681 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     7741 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     7801 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     7861 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     7921 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     7981 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     8041 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     8101 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     8161 aagtgccacc tgacgtc
//
