LOCUS       sequence-149779-        8199 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gene Addition of 3xFLAG_Twin_Strep_EZH2 to the AAVS1 Genomic Safe 
            Harbor Locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8199)
  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 8199)
  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_EZH2
FEATURES             Location/Qualifiers
     source          1..8199
                     /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    5052..5276
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    5296..6132
                     /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(6209..6226)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6380..6968)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6460..6479)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7139..7930,7931..7999))
                     /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     7762..7781
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8000..8104)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8172..8190
                     /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 aaagggcgcc ggccagactg ggaagaaatc tgagaaggga
     2821 ccagtttgtt ggcggaagcg tgtaaaatca gagtacatgc gactgagaca gctcaagagg
     2881 ttcagacgag ctgatgaagt aaagagtatg tttagttcca atcgtcagaa aattttggaa
     2941 agaacggaaa tcttaaacca agaatggaaa cagcgaagga tacagcctgt gcacatcctg
     3001 acttctgtga gctcattgcg cgggactagg gagtgttcgg tgaccagtga cttggatttt
     3061 ccaacacaag tcatcccatt aaagactctg aatgcagttg cttcagtacc cataatgtat
     3121 tcttggtctc ccctacagca gaattttatg gtggaagatg aaactgtttt acataacatt
     3181 ccttatatgg gagatgaagt tttagatcag gatggtactt tcattgaaga actaataaaa
     3241 aattatgatg ggaaagtaca cggggataga gaatgtgggt ttataaatga tgaaattttt
     3301 gtggagttgg tgaatgccct tggtcaatat aatgatgatg acgatgatga tgatggagac
     3361 gatcctgaag aaagagaaga aaagcagaaa gatctggagg atcaccgaga tgataaagaa
     3421 agccgcccac ctcggaaatt tccttctgat aaaatttttg aagccatttc ctcaatgttt
     3481 ccagataagg gcacagcaga agaactaaag gaaaaatata aagaactcac cgaacagcag
     3541 ctcccaggcg cacttcctcc tgaatgtacc cccaacatag atggaccaaa tgctaaatct
     3601 gttcagagag agcaaagctt acactccttt catacgcttt tctgtaggcg atgttttaaa
     3661 tatgactgct tcctacatcg taagtgcaat tattcttttc atgcaacacc caacacttat
     3721 aagcggaaga acacagaaac agctctagac aacaaacctt gtggaccaca gtgttaccag
     3781 catttggagg gagcaaagga gtttgctgct gctctcaccg ctgagcggat aaagacccca
     3841 ccaaaacgtc caggaggccg cagaagagga cggcttccca ataacagtag caggcccagc
     3901 acccccacca ttaatgtgct ggaatcaaag gatacagaca gtgataggga agcagggact
     3961 gaaacggggg gagagaacaa tgataaagaa gaagaagaga agaaagatga aacttcgagc
     4021 tcctctgaag caaattctcg gtgtcaaaca ccaataaaga tgaagccaaa tattgaacct
     4081 cctgagaatg tggagtggag tggtgctgaa gcctcaatgt ttagagtcct cattggcact
     4141 tactatgaca atttctgtgc cattgctagg ttaattggga ccaaaacatg tagacaggtg
     4201 tatgagttta gagtcaaaga atctagcatc atagctccag ctcccgctga ggatgtggat
     4261 actcctccaa ggaaaaagaa gaggaaacac cggttgtggg ctgcacactg cagaaagata
     4321 cagctgaaaa aggacggctc ctctaaccat gtttacaact atcaaccctg tgatcatcca
     4381 cggcagcctt gtgacagttc gtgcccttgt gtgatagcac aaaatttttg tgaaaagttt
     4441 tgtcaatgta gttcagagtg tcaaaaccgc tttccgggat gccgctgcaa agcacagtgc
     4501 aacaccaagc agtgcccgtg ctacctggct gtccgagagt gtgaccctga cctctgtctt
     4561 acttgtggag ccgctgacca ttgggacagt aaaaatgtgt cctgcaagaa ctgcagtatt
     4621 cagcggggct ccaaaaagca tctattgctg gcaccatctg acgtggcagg ctgggggatt
     4681 tttatcaaag atcctgtgca gaaaaatgaa ttcatctcag aatactgtgg agagattatt
     4741 tctcaagatg aagctgacag aagagggaaa gtgtatgata aatacatgtg cagctttctg
     4801 ttcaacttga acaatgattt tgtggtggat gcaacccgca agggtaacaa aattcgtttt
     4861 gcaaatcatt cggtaaatcc aaactgctat gcaaaagtta tgatggttaa cggtgatcac
     4921 aggataggta tttttgccaa gagagccatc cagactggcg aagagctgtt ttttgattac
     4981 agatacagcc aggctgatgc cctgaagtat gtcggcatcg aaagagaaat ggaaatccct
     5041 tgacctgcag gctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     5101 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     5161 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     5221 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgggtcg
     5281 acagtactaa gctttactag ggacaggatt ggtgacagaa aagccccatc cttaggcctc
     5341 ctccttccta gtctcctgat attgggtcta acccccacct cctgttaggc agattcctta
     5401 tctggtgaca cacccccatt tcctggagcc atctctctcc ttgccagaac ctctaaggtt
     5461 tgcttacgat ggagccagag aggatcctgg gagggagagc ttggcagggg gtgggaggga
     5521 agggggggat gcgtgacctg cccggttctc agtggccacc ctgcgctacc ctctcccaga
     5581 acctgagctg ctctgacgcg gctgtctggt gcgtttcact gatcctggtg ctgcagcttc
     5641 cttacacttc ccaagaggag aagcagtttg gaaaaacaaa atcagaataa gttggtcctg
     5701 agttctaact ttggctcttc acctttctag tccccaattt atattgttcc tccgtgcgtc
     5761 agttttacct gtgagataag gccagtagcc agccccgtcc tggcagggct gtggtgagga
     5821 ggggggtgtc cgtgtggaaa actccctttg tgagaatggt gcgtcctagg tgttcaccag
     5881 gtcgtggccg cctctactcc ctttctcttt ctccatcctt ctttccttaa agagtcccca
     5941 gtgctatctg ggacatattc ctccgcccag agcagggtcc cgcttcccta aggccctgct
     6001 ctgggcttct gggtttgagt ccttggcaag cccaggagag gcgctcaggc ttccctgtcc
     6061 cccttcctcg tccaccatct catgcccctg gctctcctgc cccttcccta caggggttcc
     6121 tggctctgct ctaagggcga attctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     6181 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     6241 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     6301 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     6361 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     6421 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     6481 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     6541 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     6601 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     6661 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     6721 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     6781 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     6841 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     6901 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     6961 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     7021 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     7081 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     7141 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     7201 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7261 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     7321 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     7381 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     7441 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     7501 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     7561 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     7621 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     7681 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     7741 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     7801 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     7861 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     7921 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     7981 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     8041 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     8101 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     8161 taacctataa aaataggcgt atcacgaggc cctttcgtc
//
