LOCUS       sequence-180428-        6663 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Doyon lab Tandem-Affinity Purification Following Nuclease-Driven 
            Gene Addition to the AAVS1 Genomic Safe Harbor Locus. Transgene 
            expression is controlled by an Auto-Regulated Tet-On 3G System..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6663)
  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 6663)
  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_Tet3G_3xFLAG_Twin_Strep
FEATURES             Location/Qualifiers
     source          1..6663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    123..926
                     /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    933..958
                     /label=SA
                     /note="splice acceptor site"
     CDS             982..1035
                     /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             1039..1635
                     /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     1532..1552
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1651..1772
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1662..1681)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1716..1735
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1841..2587)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     promoter        2602..3052
                     /label=TRE3G BI promoter
                     /note="3rd-generation Tet-responsive bidirectional promoter
                     that can be activated by binding of Tet-On(R) 3G"
     protein_bind    2682..2700
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2718..2736
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2754..2772
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2790..2808
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2826..2844
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2862..2880
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2898..2916
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3080..3100
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             3119..3184
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             join(3194..3199,3200..3223,3224..3259,3260..3283)
                     /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    3319..3543
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    3563..4399
                     /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(4476..4493)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4647..5235)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4727..4746)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5406..6197,6198..6266))
                     /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     6029..6048
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6267..6371)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6439..6457
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6495..6517)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6617..6636
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc attcaggctg
       61 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gaattcgccc
      121 tttgctttct ctgaccagca ttctctcccc tgggcctgtg ccgctttctg tctgcagctt
      181 gtggcctggg tcacctctac ggctggccca gatccttccc tgccgcctcc ttcaggttcc
      241 gtcttcctcc actccctctt ccccttgctc tctgctgtgt tgctgcccaa ggatgctctt
      301 tccggagcac ttccttctcg gcgctgcacc acgtgatgtc ctctgagcgg atcctccccg
      361 tgtctgggtc ctctccgggc atctctcctc cctcacccaa ccccatgccg tcttcactcg
      421 ctgggttccc ttttccttct ccttctgggg cctgtgccat ctctcgtttc ttaggatggc
      481 cttctccgac ggatgtctcc cttgcgtccc gcctcccctt cttgtaggcc tgcatcatca
      541 ccgtttttct ggacaacccc aaagtacccc gtctccctgg ctttagccac ctctccatcc
      601 tcttgctttc tttgcctgga caccccgttc tcctgtggat tcgggtcacc tctcactcct
      661 ttcatttggg cagctcccct acccccctta cctctctagt ctgtgctagc tcttccagcc
      721 ccctgtcatg gcatcttcca ggggtccgag agctcagcta gtcttcttcc tccaacccgg
      781 gcccctatgt ccacttcagg acagcatgtt tgctgcctcc agggatcctg tgtccccgag
      841 ctgggaccac cttatattcc cagggccggt taatgtggct ctggttctgg gtacttttat
      901 ctgtcccctc caccccacag tggggcaagc ttctgacctc ttctcttcct cccacagggc
      961 ctcgagagat ctggcggcgg agagggcaga ggaagtcttc taacatgcgg tgacgtggag
     1021 gagaatcccg gccctagcac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     1081 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     1141 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     1201 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     1261 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     1321 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     1381 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     1441 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     1501 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     1561 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     1621 aagcccggtg cctgatctag acagacatga taagatacat tgatgagttt ggacaaacca
     1681 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
     1741 ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt
     1801 ttcaggttca gggggaggtg tgggaggttt tttaatgcat ttacccgggg agcatgtcaa
     1861 ggtcaaaatc gtcaagagcg tcagcaggca gcatatcaag gtcaaagtcg tcaagggcat
     1921 cggctgggag catgtctaag tcaaaatcgt caagggcgtc ggtcggcccg ccgctttcgc
     1981 actttagctg tttctccagg ccacatatga ttagttccag gccgaaaagg aaggcaggtt
     2041 cggctccctg ccggtcgaac agctcaattg cttgtttcag aagtgggggc atagaatcgg
     2101 tggtaggtgt ctctctttcc tcttttgcta cttgatgctc ctgttcctcc aatacgcagc
     2161 ccagtgtaaa gtggcccacg gcggacagag cgtacagtgc gttctccagg gagaagcctt
     2221 gctgacacag gaacgcgagc tgattttcca gggtttcgta ctgtttctct gttgggcggg
     2281 tgccgagatg cactttagcc ccgtcgcgat gtgagaggag agcacagcgg tatgacttgg
     2341 cgttgttccg cagaaagtct tgccatgact cgccttccag ggggcaggag tgggtatgat
     2401 gcctgtccag catctcgatt ggcagggcat cgagcagggc ccgcttgttc ttcacgtgcc
     2461 agtacagggt aggctgctca actcccagct tttgagcgag tttccttgtc gtcaggcctt
     2521 cgataccgac tccattgagt aattccagag cagagtttat gactttgctc ttgtccagtc
     2581 tagacatggt gaattctcca ggcgatctga cggttcacta aacgagctct gcttatatag
     2641 gcctcccacc gtacacgcca cctcgacata ctcgagttta ctccctatca gtgatagaga
     2701 acgtatgaag agtttactcc ctatcagtga tagagaacgt atgcagactt tactccctat
     2761 cagtgataga gaacgtataa ggagtttact ccctatcagt gatagagaac gtatgaccag
     2821 tttactccct atcagtgata gagaacgtat ctacagttta ctccctatca gtgatagaga
     2881 acgtatatcc agtttactcc ctatcagtga tagagaacgt ataagcttta ggcgtgtacg
     2941 gtgggcgcct ataaaagcag agctcgttta gtgaaccgtc agatcgcctg gagcaattcc
     3001 acaacacttt tgtcttatac caactttccg taccacttcc taccctcgta aaggtaccct
     3061 cagccttaag gcggccgccg agaatctgta ttttcaggga gccatgggat ccgccggcga
     3121 ctacaaggac cacgacggcg attataagga tcacgacatc gactacaaag acgacgatga
     3181 caagggcgcc agcagcgcct ggtcccaccc tcagtttgag aagggcggag gctctggcgg
     3241 cggaagcgga ggatctgctt ggagccaccc ccagttcgaa aagtgataac tcgagtctag
     3301 acgtttaaac cctgcaggct gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     3361 ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg
     3421 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     3481 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta
     3541 tgggtcgaca gtactaagct ttactaggga caggattggt gacagaaaag ccccatcctt
     3601 aggcctcctc cttcctagtc tcctgatatt gggtctaacc cccacctcct gttaggcaga
     3661 ttccttatct ggtgacacac ccccatttcc tggagccatc tctctccttg ccagaacctc
     3721 taaggtttgc ttacgatgga gccagagagg atcctgggag ggagagcttg gcagggggtg
     3781 ggagggaagg gggggatgcg tgacctgccc ggttctcagt ggccaccctg cgctaccctc
     3841 tcccagaacc tgagctgctc tgacgcggct gtctggtgcg tttcactgat cctggtgctg
     3901 cagcttcctt acacttccca agaggagaag cagtttggaa aaacaaaatc agaataagtt
     3961 ggtcctgagt tctaactttg gctcttcacc tttctagtcc ccaatttata ttgttcctcc
     4021 gtgcgtcagt tttacctgtg agataaggcc agtagccagc cccgtcctgg cagggctgtg
     4081 gtgaggaggg gggtgtccgt gtggaaaact ccctttgtga gaatggtgcg tcctaggtgt
     4141 tcaccaggtc gtggccgcct ctactccctt tctctttctc catccttctt tccttaaaga
     4201 gtccccagtg ctatctggga catattcctc cgcccagagc agggtcccgc ttccctaagg
     4261 ccctgctctg ggcttctggg tttgagtcct tggcaagccc aggagaggcg ctcaggcttc
     4321 cctgtccccc ttcctcgtcc accatctcat gcccctggct ctcctgcccc ttccctacag
     4381 gggttcctgg ctctgctcta agggcgaatt ctgcattaat gaatcggcca acgcgcgggg
     4441 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4501 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4561 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4621 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4681 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4741 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4801 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4861 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4921 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4981 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5041 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5101 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5161 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5221 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5281 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5341 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5401 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5461 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5521 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5581 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5641 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5701 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5761 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5821 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5881 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5941 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6001 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6061 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6121 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6181 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6241 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6301 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6361 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     6421 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     6481 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     6541 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     6601 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     6661 gaa
//
