LOCUS       sequence-219958-        8193 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 8193)
  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 8193)
  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..8193
                     /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"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      689..698
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             695..808
                     /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             827..847
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             854..880
                     /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             854..880
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             881..958
                     /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          3804..4033
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     4016..4035
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    4106..4678
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4272..4289)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4499..4518
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4711..5310
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4711..4730)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5207..5227
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(5446..5463)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5452..5676
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5723..5742
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5802..5818)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5802..5818)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5815..5837)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5826..5842
                     /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(5850..5856,5857..5874,5875..5880))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5895..5916
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6033..6050)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6204..6792)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6284..6303)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6963..7754,7755..7823))
                     /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     7586..7605
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7824..7928)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8003..8022)
                     /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 agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 ggtaccgagc tcggatcgat ttaattaagc caccatggac gagaagacca ccggctggcg
      721 gggcggccac gtggtggagg gcctggccgg cgagctggag cagctgcggg cccggctgga
      781 gcaccacccc cagggccagc gggagcccgg cggaagcccc ggtggcgaga acctgtactt
      841 ccagggcgca agctacccct acgacgtgcc cgactacgcc aagcggcggt ggaagaagaa
      901 cttcatcgcc gtgagcgccg ccaaccggtt caagaagatc agcagcagcg gcgccctgat
      961 tgatggggga tctggccccg gcggcggcgc gccaagtgga ggatctcaag tccacatttt
     1021 ttggggtgct ccaattgctc cactgaaaat cacagtatca gaagacacag cttctttaat
     1081 gtctgttgct gacccctgga aaaaaattca gcttttatac agtcaacatt ctttatatct
     1141 gaaggatgaa aaacagcaca aaaatcttga aaactataaa gtcccagaat ctattggttc
     1201 tccagatctt agtggtcatt tcttagcaaa ctgtatgaat agacatgttc atgtgaaaga
     1261 tgactttgta cgttctgttt ctgaaacaca gaatatagaa tcccagaaga ttcactcctc
     1321 tagactgagt gatataacta gctctaatat gcaaatatgt ggatttaaaa gcacagttcc
     1381 gcatttcacc gaagaagaaa agtatcaaaa gcttctcagt gaaaataaaa ttagagatga
     1441 acagcctaaa catcagccag atatatgtgg taagaacttt aacacaaatt tgtttcagtt
     1501 gggccataaa tgtgcagctg tgttggattt ggtttgtagt actgaaaaaa ttaatatagg
     1561 gcctgaagtg gtacaaagag agtgtgtgcc aacagaatat catgaaatac aaaaccagtg
     1621 tttgggatta ttttcctcga acgcagtaga taagtcaagg tctgaagcag cagttaggaa
     1681 ggtctcagac cttaaaatat caactgatac agaatttctc agtataatta cctccagcca
     1741 ggttgctttt ttagctcaaa agaaagataa aaggcggagt cctgtaaata aagggaatgt
     1801 aaacatggag actgaaccaa aggcaagtta cggggagata agaatacctg aagagaattc
     1861 gattcagctt gatggtttta cagaagcata tgaaagtgga caaaaccaag catattccct
     1921 tgaacttttt agtcctgttt gtcctaaaac agaaaatagc cgcattcaca taaactctga
     1981 taaaggtctt gaagaacata caggatctca agaacttttc agttctgaag atgaactgcc
     2041 accaaatgag atacgtattg agttgtgtag ctcaggaata ctgtgttccc aactaaatac
     2101 cttccacaaa agtgctatta aaagaagctg tacctctgaa gataaagtgg gccagtctga
     2161 agctctatct agagtccttc aagtagctaa gaaaatgaag ttgatttcta atggaggaga
     2221 ttctgctgta gaaatggatc ggagaaatgt gtctgaattt aagagtatta aaaaaacatc
     2281 attaataaaa aactgtgatt ctaaaagcca gaagtataat tgtttagtca tggtgctatc
     2341 tccatgccat gtgaaggaaa taaacataaa attcggacca aattctggct ctaaagtgcc
     2401 tttagcaaca gttacagtaa ttgatcaatc agaaactaag aagaaggttt ttctgtggag
     2461 gactgcagca ttttgggcat ttacagtgtt tcttggagat ataattttac tcacagatgt
     2521 tgttattcat gaggaccaat ggattggcga gacagtacta caatcaacat ttagcagtca
     2581 gttattaaat cttgggagtt attcatctat tcagcctgaa gaatattcca gtgtagttag
     2641 tgaagttgta cttcaagact tactggcata tgtgtcctca aaacattcct acctcagaga
     2701 tcttcctccg aggcagcctc agagggtgaa cagtatagac tttgtagaat tggagcacct
     2761 tcaacctgat gtattagtcc acgcagtact aagagttgtt gatttcacta tactgacaga
     2821 ggcagtatac agttatagag gacagaagca gaaaaaagtt atgttaacag tggaacaggc
     2881 ccaagatcaa cattatgcgc ttgtattatg gggtcctgga gcagcctggt accctcaact
     2941 tcaaaggaaa aaaggttata tttgggaatt taaatatctt tttgttcagt gcaattacac
     3001 actagaaaac ctagaattgc atacaacgcc ttggtcatcc tgtgagtgct tgtttgatga
     3061 tgatataagg gcaattacat ttaaagcaaa atttcaaaaa agtgcaccct cctttgtgaa
     3121 gatatcagac ttagcaaccc acctagagga taagtgttca ggagtggttc tgattaaagc
     3181 ccagatttca gagctggcat ttcctattac agcatctcag aagatagcgc taaatgctca
     3241 cagttctctg aagagtattt tttcttctct tcccaacatc gtatatactg gttgtgcaaa
     3301 atgtggattg gaactagaaa cagatgagaa caggatctac aaacaatgtt ttagctgctt
     3361 gccatttact atgaagaaaa tatattatag gccagcgtta atgactgcca ttgatggaag
     3421 acatgatgtt tgtatccgtg tagaatcaaa gctgatagag aagattcttc tcaacatttc
     3481 tgcagactgc ctcaacagag tgatagttcc ttcctcagag atcacctatg ggatggtcgt
     3541 ggcagacctg ttccactcct tgttggcagt cagcgcagaa ccttgtgtat taaagattca
     3601 gagccttttt gtgttagatg aaaacagcta tccattacaa caagatttct ccctcctgga
     3661 tttttatcct gacattgtaa agcatggagc caatgcccgt ctctgagcgg ccgcgtcgac
     3721 ggaattcgcc cttttcggat ccactagtaa cggccgccag tgtgctggaa ttaattcgct
     3781 gtctgcgagg gccagctgtt ggggtgagta ctccctctca aaagcgggca tgacttctgc
     3841 gctaagattg tcagtttcca aaaacgagga ggatttgata ttcacctggc ccgcggtgat
     3901 gcctttgagg gtggccgcgt ccatctggtc agaaaagaca atctttttgt tgtcaagctt
     3961 gaggtgtggc aggcttgaga tctggccata cacttgagtg acaatgacat ccactttgcc
     4021 tttctctcca caggtgtcca ctcccaggtc caactgcagg tcgagcatgc atctagggcg
     4081 gccaattccg cccctctccc ccccccccct ctcctccccc ccccctaacg ttactggccg
     4141 aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc
     4201 gtcttttggc aatgtgaggg cccggaaacc tggccctgtc ttcttgacga gcattcctag
     4261 gggtctttcc cctctcgcca aaggaatgca aggtctgttg aatgtcgtga aggaagcagt
     4321 tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg accctttgca ggcagcggaa
     4381 ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca cgtgtataag atacacctgc
     4441 aaaggcggca caaccccagt gccacgttgt gagttggata gttgtggaaa gagtcaaatg
     4501 gctctcctca agcgtattca acaaggggct gaaggatgcc cagaaggtac cccattgtat
     4561 gggatctgat ctggggcctc ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa
     4621 acgtctaggc cccccgaacc acggggacgt ggttttcctt tgaaaaacac gatgataagc
     4681 ttgccacaac ccacaaggag acgaccttcc atgaccgagt acaagcccac ggtgcgcctc
     4741 gccacccgcg acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac
     4801 cccgccacgc gccacaccgt cgacccggac cgccacatcg agcgggtcac cgagctgcaa
     4861 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     4921 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     4981 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     5041 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     5101 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     5161 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     5221 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     5281 tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc
     5341 gaaaggagcg cacgacccca tggctccgac cgaagccacc cggggcggcc ccgccgaccc
     5401 cgcacccgcc cccgaggccc accgactcta gagctcgctg atcagcctcg actgtgcctt
     5461 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     5521 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     5581 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     5641 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     5701 ggggctcgag tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     5761 tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     5821 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     5881 gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     5941 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6001 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     6061 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     6121 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     6181 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     6241 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     6301 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     6361 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     6421 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     6481 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     6541 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     6601 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     6661 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     6721 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     6781 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     6841 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     6901 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     6961 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     7021 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     7081 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     7141 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     7201 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     7261 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     7321 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     7381 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     7441 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     7501 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     7561 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     7621 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     7681 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     7741 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     7801 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     7861 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     7921 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg
     7981 atcccctatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc
     8041 tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa
     8101 caaggcaagg cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc
     8161 tgcttcgcga tgtacgggcc agatatacgc gtt
//
