LOCUS       sequence-418112-        5653 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 5653)
  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 5653)
  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: pJT311 AAV-Rapamycin Inducible-NZF-Citrine
FEATURES             Location/Qualifiers
     source          1..5653
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(156..175)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      969..1557
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1458..1477
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   1619..1748
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             1897..1923
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             2653..2706
                     /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             2707..2733
                     /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(2740..2913,2914..2919,2920..3102)
                     /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             3109..3432
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             3109..3429
                     /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        3634..3747
                     /label=minimal IL-2 promoter
                     /label=IL-2 promoter
                     /note="minimal promoter from the human interleukin-2 gene"
     regulatory      3751..3760
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3757..3759,3760..3762,3763..4473)
                     /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(3802..3823)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4063..4082)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4410..4431
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     repeat_region   4546..4686
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4546..4675
                     /label=AAV2 ITR
     rep_origin      4761..5216
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4848..4867)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5058..5079
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5233..5252)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5346..5368
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5400..5418)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5486..5590
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5591..5653,1..6,7..798)
                     /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"
ORIGIN
        1 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
       61 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
      121 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
      181 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
      241 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
      301 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
      361 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
      421 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
      481 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
      541 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
      601 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
      661 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
      721 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
      781 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
      841 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
      901 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
      961 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     1021 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     1081 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     1141 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     1201 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     1261 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     1321 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     1381 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     1441 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     1501 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     1561 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgtcc
     1621 tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg
     1681 tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag
     1741 gggttcctgc ggccgcacgc gttaatgatg ggcgctacag taatgatggg cggaattcct
     1801 cgaggtactt atataagggg gtgggggcgc gttcgtcctc agtcgcgatc gaacactcga
     1861 gccgagcaga cgtgcctaac cggtgccgcc accatgcctg ctgccaagag ggtcaagttg
     1921 gactggcatg aaggcctgga agaggcatct cgtttgtact ttggggaaag gaacgtgaaa
     1981 ggcatgtttg aggtgctgga gcccttgcat gctatgatgg aacggggccc ccagactctg
     2041 aaggaaacat cttttaatca ggcctatggt cgtgatttaa tggaggccca agagtggtgc
     2101 aggaagtaca tgaaatcagg gaatgtcaag gacctcaccc aagcctggga cctctattat
     2161 catgtgttcc gacgaatctc aaagcagtct ggatccggat caggctccga gggccagagc
     2221 gacgagagag ccctgctgga ccagctgcac accctgctga gcaacaccga cgccaccggc
     2281 ctggaggaga tcgacagagc cctgggcatc cccgagctgg tgaaccaggg ccaggccctg
     2341 gagcccaagc agggaggagg aggtagcgga ttcgtgaccc tgaaggacgt gggcatggac
     2401 ttcaccctgg gcgactggga gcagctgggc ctggagcagg gcgacacctt ctgggacacc
     2461 gccctggaca actgccagga cctgttcctg ctggaccccc ccggaggagg aggtagcgga
     2521 cacgagaagt tccccagcga cctggacctg gacatgttca acggcagcct ggagtgcgac
     2581 atggagagca tcatcagaag cgagctgatg gacgccgacg gcctggactt caacttcgac
     2641 agcggatctg gagagggaag gggaagcctc ctaacttgcg gagatgtcga ggagaatcct
     2701 ggcccacctg ccgctaagcg tgtcaagttg gactctagag aacgcccgta tgcttgccct
     2761 gtcgagtcct gcgatcgccg cttttctcgc tcggatgagc ttacccgcca tatccgcatc
     2821 cacacaggcc agaagccctt ccagtgtcga atctgcatgc gtaacttcag tcgtagtgac
     2881 caccttacca cccacatccg cacccacaca ggcggcggcc gcaggaggaa gaaacgcacc
     2941 agcatagaga ctaacatccg tgtggcctta gagaagagtt tcttggagaa tcaaaagcct
     3001 acctcggaag agatcactat gattgctgat cagctcaata tggaaaaaga ggtgattcgt
     3061 gtttggttct gtaaccgccg ccagaaagaa aaaagaatca acaagggcgg cgtgcaggtg
     3121 gagactatct ccccaggcga cgggcgcacc ttccccaagc gcggccagac ctgcgtggtg
     3181 cactacaccg ggatgcttga agatggaaag aaatttgatt cctcccggga cagaaacaag
     3241 ccctttaagt ttatgctagg caagcaggag gtgatccgag gctgggaaga aggggttgcc
     3301 cagatgagtg tgggtcagag agccaaactg actatatctc cagattatgc ctatggtgcc
     3361 actgggcacc caggcatcat cccaccacat gccactctcg tctttgatgt ggagcttcta
     3421 aaactggaat aacatatgaa ataaaatacg aaatgcgtac gtaatgatgg gcgctaaatt
     3481 aatgatgggc ggagttgtaa tgatgggcgc tcgtttaatg atgggcgtct agctaatgat
     3541 gggcgggcga ttaatgatgg gcgaaggact aatgatgggc gcagcggtaa tgatgggcga
     3601 tcagctaatg atgggcgacg cgaattaatt caacattttg acacccccat aatatttttc
     3661 cagaattaac agtataaatt gcatctcttg ttcaagagtt ccctatcact ctctttaatc
     3721 actactcaca gtaacctcaa ctcctgcgcc gccaccatgg tgagcaaggg cgaggagctg
     3781 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     3841 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     3901 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc
     3961 ctgatgtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     4021 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     4081 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     4141 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     4201 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     4261 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     4321 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagctacca gtccgccctg
     4381 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     4441 gggatcactc tcggcatgga cgagctgtac aagtaagcct aataaagagc tcagatgcat
     4501 gcatcagagt gtgttggttt tttgtgtgtc ggaccgagcg gccgcaggaa cccctagtga
     4561 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     4621 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     4681 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4741 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     4801 ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc ctttcgcttt
     4861 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     4921 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     4981 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     5041 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca actctatctc
     5101 gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt taaaaaatga
     5161 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     5221 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     5281 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     5341 tgtgaccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcaaaggg
     5401 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     5461 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5521 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5581 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     5641 ttgccttcct gtt
//
