LOCUS       sequence-418116-        6157 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 6157)
  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 6157)
  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: pJT365 AAV-Rapamycin Inducible-S3H
FEATURES             Location/Qualifiers
     source          1..6157
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      635..1223
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1124..1143
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1285..1414
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             1563..1589
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             1875..2417
                     /codon_start=1
                     /product="C-terminal portion of the p65 subunit of mouse 
                     NF-kappa-B"
                     /label=p65
                     /translation="PSGQISNQALALAPSSAPVLAQTMVPSSAMVPLAQPPAPAPVLTP
                     GPPQSLSAPVPKSTQAGEGTLSEALLHLQFDADEDLGALLGNSTDPGVFTDLASVDNSE
                     FQQLLNQGVSMSHSTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGTSGLPNGLSGDED
                     FSSIADMDFSALLSQISS"
     CDS             2442..2813
                     /codon_start=1
                     /product="C-terminal activation domain from the human heat 
                     shock transcription factor HSF1"
                     /label=HSF1
                     /translation="GFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQELLSPQEPPR
                     PPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGDGFAED
                     PTISLLTGSEPPKAKDPTVS"
     CDS             2823..2876
                     /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             2877..2903
                     /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(2910..3083,3084..3089,3090..3272)
                     /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             3279..3602
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             3279..3599
                     /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        3804..3917
                     /label=minimal IL-2 promoter
                     /label=IL-2 promoter
                     /note="minimal promoter from the human interleukin-2 gene"
     regulatory      3921..3930
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3927..3929,3930..3932,3933..4643)
                     /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(3972..3993)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4233..4252)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4580..4601
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   4716..4856
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4716..4845
                     /label=AAV2 ITR
     rep_origin      4931..5386
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5018..5037)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5228..5249
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5403..5422)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5516..5538
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5570..5588)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5656..5760
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5761..5829,5830..6157,1..464)
                     /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(5979..5998)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
       61 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
      121 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
      181 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
      241 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
      301 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
      361 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
      421 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
      481 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
      541 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
      601 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
      661 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
      721 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
      781 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
      841 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
      901 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
      961 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     1021 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     1081 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     1141 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     1201 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     1261 tttgctggcc ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg
     1321 ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga
     1381 gagggagtgg ccaactccat cactaggggt tcctgcggcc gcacgcgtta atgatgggcg
     1441 ctacagtaat gatgggcgga attcctcgag gtacttatat aagggggtgg gggcgcgttc
     1501 gtcctcagtc gcgatcgaac actcgagccg agcagacgtg cctaaccggt gccgccacca
     1561 tgcctgctgc caagagggtc aagttggact ggcatgaagg cctggaagag gcatctcgtt
     1621 tgtactttgg ggaaaggaac gtgaaaggca tgtttgaggt gctggagccc ttgcatgcta
     1681 tgatggaacg gggcccccag actctgaagg aaacatcttt taatcaggcc tatggtcgtg
     1741 atttaatgga ggcccaagag tggtgcagga agtacatgaa atcagggaat gtcaaggacc
     1801 tcacccaagc ctgggacctc tattatcatg tgttccgacg aatctcaaag cagtctggat
     1861 ccggatcagg ctccccttca gggcagatca gcaaccaggc cctggctctg gcccctagct
     1921 ccgctccagt gctggcccag actatggtgc cctctagtgc tatggtgcct ctggcccagc
     1981 cacctgctcc agcccctgtg ctgaccccag gaccacccca gtcactgagc gctccagtgc
     2041 ccaagtctac acaggccggc gaggggactc tgagtgaagc tctgctgcac ctgcagttcg
     2101 acgctgatga ggacctggga gctctgctgg ggaacagcac cgatcccgga gtgttcacag
     2161 atctggcctc cgtggacaac tctgagtttc agcagctgct gaatcagggc gtgtccatgt
     2221 ctcatagtac agccgaacca atgctgatgg agtaccccga agccattacc cggctggtga
     2281 ccggcagcca gcggcccccc gaccccgctc caactcccct gggaaccagc ggcctgccta
     2341 atgggctgtc cggagatgaa gacttctcaa gcatcgctga tatggacttt agtgccctgc
     2401 tgtcacagat ttcctctagt gggcagggag gaggtggaag cggcttcagc gtggacacca
     2461 gtgccctgct ggacctgttc agcccctcgg tgaccgtgcc cgacatgagc ctgcctgacc
     2521 ttgacagcag cctggccagt atccaagagc tcctgtctcc ccaggagccc cccaggcctc
     2581 ccgaggcaga gaacagcagc ccggattcag ggaagcagct ggtgcactac acagcgcagc
     2641 cgctgttcct gctggacccc ggctccgtgg acaccgggag caacgacctg ccggtgctgt
     2701 ttgagctggg agagggctcc tacttctccg aaggggacgg cttcgccgag gaccccacca
     2761 tctccctgct gacaggctcg gagcctccca aagccaagga ccccactgtc tccggatctg
     2821 gagagggaag gggaagcctc ctaacttgcg gagatgtcga ggagaatcct ggcccacctg
     2881 ccgctaagcg tgtcaagttg gactctagag aacgcccgta tgcttgccct gtcgagtcct
     2941 gcgatcgccg cttttctcgc tcggatgagc ttacccgcca tatccgcatc cacacaggcc
     3001 agaagccctt ccagtgtcga atctgcatgc gtaacttcag tcgtagtgac caccttacca
     3061 cccacatccg cacccacaca ggcggcggcc gcaggaggaa gaaacgcacc agcatagaga
     3121 ctaacatccg tgtggcctta gagaagagtt tcttggagaa tcaaaagcct acctcggaag
     3181 agatcactat gattgctgat cagctcaata tggaaaaaga ggtgattcgt gtttggttct
     3241 gtaaccgccg ccagaaagaa aaaagaatca acaagggcgg cgtgcaggtg gagactatct
     3301 ccccaggcga cgggcgcacc ttccccaagc gcggccagac ctgcgtggtg cactacaccg
     3361 ggatgcttga agatggaaag aaatttgatt cctcccggga cagaaacaag ccctttaagt
     3421 ttatgctagg caagcaggag gtgatccgag gctgggaaga aggggttgcc cagatgagtg
     3481 tgggtcagag agccaaactg actatatctc cagattatgc ctatggtgcc actgggcacc
     3541 caggcatcat cccaccacat gccactctcg tctttgatgt ggagcttcta aaactggaat
     3601 aacatatgaa ataaaatacg aaatgcgtac gtaatgatgg gcgctaaatt aatgatgggc
     3661 ggagttgtaa tgatgggcgc tcgtttaatg atgggcgtct agctaatgat gggcgggcga
     3721 ttaatgatgg gcgaaggact aatgatgggc gcagcggtaa tgatgggcga tcagctaatg
     3781 atgggcgacg cgaattaatt caacattttg acacccccat aatatttttc cagaattaac
     3841 agtataaatt gcatctcttg ttcaagagtt ccctatcact ctctttaatc actactcaca
     3901 gtaacctcaa ctcctgcgcc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     3961 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     4021 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     4081 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc ctgatgtgct
     4141 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     4201 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     4261 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     4321 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     4381 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     4441 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     4501 gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg agcaaagacc
     4561 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     4621 tcggcatgga cgagctgtac aagtaagcct aataaagagc tcagatgcat gcatcagagt
     4681 gtgttggttt tttgtgtgtc ggaccgagcg gccgcaggaa cccctagtga tggagttggc
     4741 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4801 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg
     4861 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     4921 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4981 agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc
     5041 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5101 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     5161 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5221 tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc gggctattct
     5281 tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga gctgatttaa
     5341 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     5401 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     5461 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccggg
     5521 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcaaaggg cctcgtgata
     5581 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     5641 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5701 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5761 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5821 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5881 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5941 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     6001 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     6061 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     6121 tgcagtgctg ccataaccat gagtgataac actgcgg
//
