LOCUS       sequence-418118-        5350 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Inducible AAV activator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5350)
  AUTHORS   Tycko J, Van MV, Aradhana, DelRosso N, Ye H, Yao D, Valbuena R, 
            Vaughan-Jackson A, Xu X, Ludwig C, Spees K, Liu K, Gu M, Khare V, 
            Mukund AX, Suzuki PH, Arana S, Zhang C, Du PP, Ornstein TS, Hess GT,
            Kamber RA, Qi LS, Khalil AS, Bintu L, Bassik MC
  TITLE     Development of compact transcriptional effectors using 
            high-throughput measurements in diverse contexts.
  JOURNAL   Nat Biotechnol. 2024 Nov 1. doi: 10.1038/s41587-024-02442-6.
  PUBMED    39487265
REFERENCE   2  (bases 1 to 5350)
  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: "Nat 
            Biotechnol. 2024 Nov 1. doi: 10.1038/s41587-024-02442-6."
COMMENT     Sequence Label: pJT315 AAV-Rapamycin Inducible-ZNF473KRAB-Citrine
FEATURES             Location/Qualifiers
     source          1..5350
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      652..1240
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1141..1160
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1302..1431
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             1580..1606
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             2033..2086
                     /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             2087..2113
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             join(2120..2293,2294..2299,2300..2482)
                     /codon_start=1
                     /product="composite human DNA-binding domain composed of 
                     domains from the transcription factors Zif268 and Oct-1 
                     (Pomerantz et al., 1995)"
                     /label=ZFHD1
                     /label=ZFHD1 (DNA binding domain)
                     /translation="ERPYACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSR
                     SDHLTTHIRTHTGGGRRRKKRTSIETNIRVALEKSFLENQKPTSEEITMIADQLNMEKE
                     VIRVWFCNRRQKEKRIN"
     CDS             2489..2812
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             2489..2809
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=DmrB
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     promoter        3014..3127
                     /label=minimal IL-2 promoter
                     /label=IL-2 promoter
                     /note="minimal promoter from the human interleukin-2 gene"
     regulatory      3131..3140
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3137..3139,3140..3142,3143..3853)
                     /codon_start=1
                     /product="enhanced variant of YFP (Heikal et al., 2001)"
                     /label=Citrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLMCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3182..3203)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3443..3462)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3790..3811
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   3926..4066
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3926..4055
                     /label=AAV2 ITR
     rep_origin      4141..4596
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4228..4247)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4438..4459
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4613..4632)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4726..4748
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4780..4798)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4866..4970
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4971..5039,5040..5350,1..481)
                     /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     complement(5189..5208)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
       61 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
      121 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
      181 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
      241 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
      301 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
      361 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
      421 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
      481 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
      541 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
      601 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
      661 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      721 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      781 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      841 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      901 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      961 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     1021 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1081 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1141 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1201 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1261 tttacggttc ctggcctttt gctggccttt tgctcacatg tcctgcaggc agctgcgcgc
     1321 tcgctcgctc actgaggccg cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga
     1381 gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc tgcggccgca
     1441 cgcgttaatg atgggcgcta cagtaatgat gggcggaatt cctcgaggta cttatataag
     1501 ggggtggggg cgcgttcgtc ctcagtcgcg atcgaacact cgagccgagc agacgtgcct
     1561 aaccggtgcc gccaccatgc ctgctgccaa gagggtcaag ttggactggc atgaaggcct
     1621 ggaagaggca tctcgtttgt actttgggga aaggaacgtg aaaggcatgt ttgaggtgct
     1681 ggagcccttg catgctatga tggaacgggg cccccagact ctgaaggaaa catcttttaa
     1741 tcaggcctat ggtcgtgatt taatggaggc ccaagagtgg tgcaggaagt acatgaaatc
     1801 agggaatgtc aaggacctca cccaagcctg ggacctctat tatcatgtgt tccgacgaat
     1861 ctcaaagcag tctggatccg gatcaggctc cttcgtgacc ctgaaggacg tgggcatgga
     1921 cttcaccctg ggcgactggg agcagctggg cctggagcag ggcgacacct tctgggacac
     1981 cgccctggac aactgccagg acctgttcct gctggacccc cccggatctg gagagggaag
     2041 gggaagcctc ctaacttgcg gagatgtcga ggagaatcct ggcccacctg ccgctaagcg
     2101 tgtcaagttg gactctagag aacgcccgta tgcttgccct gtcgagtcct gcgatcgccg
     2161 cttttctcgc tcggatgagc ttacccgcca tatccgcatc cacacaggcc agaagccctt
     2221 ccagtgtcga atctgcatgc gtaacttcag tcgtagtgac caccttacca cccacatccg
     2281 cacccacaca ggcggcggcc gcaggaggaa gaaacgcacc agcatagaga ctaacatccg
     2341 tgtggcctta gagaagagtt tcttggagaa tcaaaagcct acctcggaag agatcactat
     2401 gattgctgat cagctcaata tggaaaaaga ggtgattcgt gtttggttct gtaaccgccg
     2461 ccagaaagaa aaaagaatca acaagggcgg cgtgcaggtg gagactatct ccccaggcga
     2521 cgggcgcacc ttccccaagc gcggccagac ctgcgtggtg cactacaccg ggatgcttga
     2581 agatggaaag aaatttgatt cctcccggga cagaaacaag ccctttaagt ttatgctagg
     2641 caagcaggag gtgatccgag gctgggaaga aggggttgcc cagatgagtg tgggtcagag
     2701 agccaaactg actatatctc cagattatgc ctatggtgcc actgggcacc caggcatcat
     2761 cccaccacat gccactctcg tctttgatgt ggagcttcta aaactggaat aacatatgaa
     2821 ataaaatacg aaatgcgtac gtaatgatgg gcgctaaatt aatgatgggc ggagttgtaa
     2881 tgatgggcgc tcgtttaatg atgggcgtct agctaatgat gggcgggcga ttaatgatgg
     2941 gcgaaggact aatgatgggc gcagcggtaa tgatgggcga tcagctaatg atgggcgacg
     3001 cgaattaatt caacattttg acacccccat aatatttttc cagaattaac agtataaatt
     3061 gcatctcttg ttcaagagtt ccctatcact ctctttaatc actactcaca gtaacctcaa
     3121 ctcctgcgcc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     3181 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     3241 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     3301 cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgatgtgct tcgcccgcta
     3361 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     3421 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     3481 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     3541 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     3601 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     3661 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3721 gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc ccaacgagaa
     3781 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3841 cgagctgtac aagtaagcct aataaagagc tcagatgcat gcatcagagt gtgttggttt
     3901 tttgtgtgtc ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct
     3961 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     4021 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg
     4081 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     4141 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     4201 ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     4261 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     4321 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     4381 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     4441 gactcttgtt ccaaactgga acaacactca actctatctc gggctattct tttgatttat
     4501 aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta
     4561 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     4621 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     4681 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccggg agctgcatgt
     4741 gtcagaggtt ttcaccgtca tcaccgaaac gcgcaaaggg cctcgtgata cgcctatttt
     4801 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     4861 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     4921 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     4981 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     5041 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     5101 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     5161 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     5221 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     5281 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     5341 ccataaccat
//
