LOCUS       sequence-418107-        5728 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 5728)
  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 5728)
  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: pJT312 AAV-Rapamycin Inducible-NFZ-Citrine wit...
FEATURES             Location/Qualifiers
     source          1..5728
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   651..791
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   662..791
                     /label=AAV2 ITR
     protein_bind    798..818
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     CDS             973..999
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             1729..1782
                     /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             1783..1809
                     /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(1816..1989,1990..1995,1996..2178)
                     /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             2185..2508
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             2185..2505
                     /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        2710..2823
                     /label=minimal IL-2 promoter
                     /label=IL-2 promoter
                     /note="minimal promoter from the human interleukin-2 gene"
     regulatory      2827..2836
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2833..2835,2836..2838,2839..3549)
                     /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(2878..2899)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3139..3158)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3486..3507
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   3641..3781
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3641..3770
                     /label=AAV2 ITR
     rep_origin      3856..4311
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3943..3962)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4153..4174
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4328..4347)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4447..4469
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4507..4525)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4593..4697
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4698..4766,4767..5558)
                     /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(4916..4935)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt cctgcaggca
      661 gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt
      721 tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac
      781 taggggttcc ttctagacaa ctttgtatag aaaagttggg taccgcggcc gcacgcgtta
      841 atgatgggcg ctacagtaat gatgggcgga attcctcgag gtacttatat aagggggtgg
      901 gggcgcgttc gtcctcagtc gcgatcgaac actcgagccg agcagacgtg cctaaccggt
      961 gccgccacca tgcctgctgc caagagggtc aagttggact ggcatgaagg cctggaagag
     1021 gcatctcgtt tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc
     1081 ttgcatgcta tgatggaacg gggcccccag actctgaagg aaacatcttt taatcaggcc
     1141 tatggtcgtg atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat
     1201 gtcaaggacc tcacccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag
     1261 cagtctggat ccggatcagg ctccgagggc cagagcgacg agagagccct gctggaccag
     1321 ctgcacaccc tgctgagcaa caccgacgcc accggcctgg aggagatcga cagagccctg
     1381 ggcatccccg agctggtgaa ccagggccag gccctggagc ccaagcaggg aggaggaggt
     1441 agcggacacg agaagttccc cagcgacctg gacctggaca tgttcaacgg cagcctggag
     1501 tgcgacatgg agagcatcat cagaagcgag ctgatggacg ccgacggcct ggacttcaac
     1561 ttcgacagcg gaggaggagg tagcggattc gtgaccctga aggacgtggg catggacttc
     1621 accctgggcg actgggagca gctgggcctg gagcagggcg acaccttctg ggacaccgcc
     1681 ctggacaact gccaggacct gttcctgctg gacccccccg gatctggaga gggaagggga
     1741 agcctcctaa cttgcggaga tgtcgaggag aatcctggcc cacctgccgc taagcgtgtc
     1801 aagttggact ctagagaacg cccgtatgct tgccctgtcg agtcctgcga tcgccgcttt
     1861 tctcgctcgg atgagcttac ccgccatatc cgcatccaca caggccagaa gcccttccag
     1921 tgtcgaatct gcatgcgtaa cttcagtcgt agtgaccacc ttaccaccca catccgcacc
     1981 cacacaggcg gcggccgcag gaggaagaaa cgcaccagca tagagactaa catccgtgtg
     2041 gccttagaga agagtttctt ggagaatcaa aagcctacct cggaagagat cactatgatt
     2101 gctgatcagc tcaatatgga aaaagaggtg attcgtgttt ggttctgtaa ccgccgccag
     2161 aaagaaaaaa gaatcaacaa gggcggcgtg caggtggaga ctatctcccc aggcgacggg
     2221 cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat
     2281 ggaaagaaat ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag
     2341 caggaggtga tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc
     2401 aaactgacta tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca
     2461 ccacatgcca ctctcgtctt tgatgtggag cttctaaaac tggaataaca tatgaaataa
     2521 aatacgaaat gcgtacgtaa tgatgggcgc taaattaatg atgggcggag ttgtaatgat
     2581 gggcgctcgt ttaatgatgg gcgtctagct aatgatgggc gggcgattaa tgatgggcga
     2641 aggactaatg atgggcgcag cggtaatgat gggcgatcag ctaatgatgg gcgacgcgaa
     2701 ttaattcaac attttgacac ccccataata tttttccaga attaacagta taaattgcat
     2761 ctcttgttca agagttccct atcactctct ttaatcacta ctcacagtaa cctcaactcc
     2821 tgcgccgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     2881 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     2941 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     3001 ccctggccca ccctcgtgac caccttcggc tacggcctga tgtgcttcgc ccgctacccc
     3061 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     3121 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     3181 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     3241 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     3301 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     3361 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     3421 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc
     3481 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     3541 ctgtacaagt aagcctaata aagagctcag atgcatgcat cagagtgtgt tggttttttg
     3601 tgtgtcggac cgagcggccg cgcatgctgg ggagggccgc aggaacccct agtgatggag
     3661 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3721 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag
     3781 gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     3841 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3901 cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc
     3961 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     4021 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     4081 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     4141 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct
     4201 attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga
     4261 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     4321 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     4381 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4441 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     4501 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     4561 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     4621 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     4681 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4741 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4801 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4861 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4921 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4981 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     5041 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     5101 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     5161 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     5221 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     5281 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5341 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5401 ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5461 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5521 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5581 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5641 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5701 accccgtaga aaagatcaaa ggatcttc
//
