LOCUS       sequence-251912-        5967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doyon lab Tandem-Affinity Purification Following (i) Nuclease-Driven
            Gene Addition to the AAVS1 Genomic Safe Harbor Locus and (ii) 
            Endogenous TAP Tagging..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5967)
  AUTHORS   Dalvai M, Loehr J, Jacquet K, Huard CC, Roques C, Herst P, Cote J, 
            Doyon Y
  TITLE     A Scalable Genome-Editing-Based Approach for Mapping Multiprotein 
            Complexes in Human Cells.
  JOURNAL   Cell Rep. 2015 Oct 7. pii: S2211-1247(15)01020-7. doi: 
            10.1016/j.celrep.2015.09.009.
  PUBMED    26456817
REFERENCE   2  (bases 1 to 5967)
  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"; journalName: "Cell Rep. 
            2015 Oct 7. pii: S2211-1247(15)01020-7. doi: 
            10.1016/j.celrep.2015.09.009."
COMMENT     Sequence Label: AAVS1_Puro_PGK1_3xFLAG_Twin_Strep
FEATURES             Location/Qualifiers
     source          1..5967
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    320..1123
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     misc_feature    1130..1155
                     /label=SA
                     /note="splice acceptor site"
     CDS             1179..1232
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1236..1832
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     1729..1749
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1848..1969
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1859..1878)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1913..1932
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2042..2552
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2471..2489
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2581..2601
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2620..2685
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(2695..2700,2701..2724,2725..2760,2761..2784)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     polyA_signal    2820..3044
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3064..3900
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     primer_bind     complement(3977..3994)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4148..4736)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4228..4247)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4907..5698,5699..5767))
                     /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     5530..5549
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5768..5872)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5940..5958
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagaa ttcgcccttt gctttctctg accagcattc tctcccctgg gcctgtgccg
      361 ctttctgtct gcagcttgtg gcctgggtca cctctacggc tggcccagat ccttccctgc
      421 cgcctccttc aggttccgtc ttcctccact ccctcttccc cttgctctct gctgtgttgc
      481 tgcccaagga tgctctttcc ggagcacttc cttctcggcg ctgcaccacg tgatgtcctc
      541 tgagcggatc ctccccgtgt ctgggtcctc tccgggcatc tctcctccct cacccaaccc
      601 catgccgtct tcactcgctg ggttcccttt tccttctcct tctggggcct gtgccatctc
      661 tcgtttctta ggatggcctt ctccgacgga tgtctccctt gcgtcccgcc tccccttctt
      721 gtaggcctgc atcatcaccg tttttctgga caaccccaaa gtaccccgtc tccctggctt
      781 tagccacctc tccatcctct tgctttcttt gcctggacac cccgttctcc tgtggattcg
      841 ggtcacctct cactcctttc atttgggcag ctcccctacc ccccttacct ctctagtctg
      901 tgctagctct tccagccccc tgtcatggca tcttccaggg gtccgagagc tcagctagtc
      961 ttcttcctcc aacccgggcc cctatgtcca cttcaggaca gcatgtttgc tgcctccagg
     1021 gatcctgtgt ccccgagctg ggaccacctt atattcccag ggccggttaa tgtggctctg
     1081 gttctgggta cttttatctg tcccctccac cccacagtgg ggcaagcttc tgacctcttc
     1141 tcttcctccc acagggcctc gagagatctg gcggcggaga gggcagagga agtcttctaa
     1201 catgcggtga cgtggaggag aatcccggcc ctagcaccga gtacaagccc acggtgcgcc
     1261 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     1321 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     1381 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     1441 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     1501 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     1561 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     1621 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     1681 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     1741 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     1801 cctggtgcat gacccgcaag cccggtgcct gatctagaca gacatgataa gatacattga
     1861 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     1921 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     1981 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aatgcatcca
     2041 cggggttggg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct
     2101 ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt
     2161 ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc
     2221 ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa
     2281 cggaagccgc acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag
     2341 cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca
     2401 gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc
     2461 tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc
     2521 tcgttgaccg aatcaccgac ctctctcccc aggggtaccc tcagccttaa ggcggccgcc
     2581 gagaatctgt attttcaggg agccatggga tccgccggcg actacaagga ccacgacggc
     2641 gattataagg atcacgacat cgactacaaa gacgacgatg acaagggcgc cagcagcgcc
     2701 tggtcccacc ctcagtttga gaagggcgga ggctctggcg gcggaagcgg aggatctgct
     2761 tggagccacc cccagttcga aaagtgataa ctcgagtcta gacgtttaaa ccctgcaggc
     2821 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2881 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2941 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3001 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atgggtcgac agtactaagc
     3061 tttactaggg acaggattgg tgacagaaaa gccccatcct taggcctcct ccttcctagt
     3121 ctcctgatat tgggtctaac ccccacctcc tgttaggcag attccttatc tggtgacaca
     3181 cccccatttc ctggagccat ctctctcctt gccagaacct ctaaggtttg cttacgatgg
     3241 agccagagag gatcctggga gggagagctt ggcagggggt gggagggaag ggggggatgc
     3301 gtgacctgcc cggttctcag tggccaccct gcgctaccct ctcccagaac ctgagctgct
     3361 ctgacgcggc tgtctggtgc gtttcactga tcctggtgct gcagcttcct tacacttccc
     3421 aagaggagaa gcagtttgga aaaacaaaat cagaataagt tggtcctgag ttctaacttt
     3481 ggctcttcac ctttctagtc cccaatttat attgttcctc cgtgcgtcag ttttacctgt
     3541 gagataaggc cagtagccag ccccgtcctg gcagggctgt ggtgaggagg ggggtgtccg
     3601 tgtggaaaac tccctttgtg agaatggtgc gtcctaggtg ttcaccaggt cgtggccgcc
     3661 tctactccct ttctctttct ccatccttct ttccttaaag agtccccagt gctatctggg
     3721 acatattcct ccgcccagag cagggtcccg cttccctaag gccctgctct gggcttctgg
     3781 gtttgagtcc ttggcaagcc caggagaggc gctcaggctt ccctgtcccc cttcctcgtc
     3841 caccatctca tgcccctggc tctcctgccc cttccctaca ggggttcctg gctctgctct
     3901 aagggcgaat tctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3961 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4021 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4081 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4141 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4201 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4261 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4321 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4381 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4441 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4501 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4561 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4621 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4681 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4741 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4801 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4861 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4921 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4981 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5041 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5101 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5161 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5221 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5281 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5341 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5401 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5461 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5521 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5581 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5641 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5701 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5761 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5821 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5881 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     5941 ataggcgtat cacgaggccc tttcgtc
//
