LOCUS       sequence-369804-        4627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rapamycin inducible genetic circuit with cloning site for activator 
            domains.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4627)
  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 4627)
  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: pJT309 RapInducible1-ActivatorCloneSite-Citrine
FEATURES             Location/Qualifiers
     source          1..4627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..72)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(56..72)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(69..91)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     rep_origin      complement(244..832)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(324..343)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(968..1783)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(968..1783)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1691..1710
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1775..1794
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     1904..1920
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1904..1920
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     terminator      1921..1967
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     2081..2103
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2095..2112
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2096..2112
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2315..2341
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             2657..2710
                     /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             2711..2737
                     /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(2744..2917,2918..2923,2924..3106)
                     /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             3113..3436
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             3113..3433
                     /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        3692..3805
                     /label=minimal IL-2 promoter
                     /label=IL-2 promoter
                     /note="minimal promoter from the human interleukin-2 gene"
     regulatory      3809..3818
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3815..3817,3818..3820,3821..4531)
                     /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(3860..3881)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4121..4140)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4468..4489
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ggctaggtgg aggctcagtg atgataagtc tgcgatggtg gatgcatgtg tcatggtcat
       61 agctgtttcc tgtgtgaaat tgttatccgc tcagagggca caatcctatt ccgcgctatc
      121 cgacaatctc caagacatta ggtggagttc agttcggcgt atggcatatg tcgctggaaa
      181 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      241 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      301 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
      361 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
      421 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
      481 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
      541 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
      601 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
      661 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
      721 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
      781 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
      841 tttgatcttt tctacggggt ctgacgctct attcaacaaa gccgccgtcc cgtcaagtca
      901 gcgtaaatgg gtagggggct tcaaatcgtc cgctctgcca gtgttacaac caattaacaa
      961 attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat
     1021 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     1081 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     1141 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     1201 tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa
     1261 caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc
     1321 gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag
     1381 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     1441 caggatattc ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc
     1501 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     1561 gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     1621 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     1681 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     1741 atcgcggcct cgagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac
     1801 tgtttatgta agcagacagt tttattgttc atgatgatat atttttatct tgtgcaatgt
     1861 aacatcagag attttgagac acaacgtggc tttcccccgc cgctctagaa ctagtggatc
     1921 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     1981 cattatacga gacgtccagg ttgggatacc tgaaacaaaa cccatcgtac ggccaaggaa
     2041 gtctccaata actgtgatcc accacaagcg ccagggtttt cccagtcacg acgttgtaaa
     2101 acgacggcca gtcatgcata atccgcacgc atctggaata aggaagtgcc attccgcctg
     2161 accttgatcg atgcacgcgt taatgatggg cgctacagta atgatgggcg gaattcctcg
     2221 aggtacttat ataagggggt gggggcgcgt tcgtcctcag tcgcgatcga acactcgagc
     2281 cgagcagacg tgcctaaccg gtgccgccac catgcctgct gccaagaggg tcaagttgga
     2341 ctggcatgaa ggcctggaag aggcatctcg tttgtacttt ggggaaagga acgtgaaagg
     2401 catgtttgag gtgctggagc ccttgcatgc tatgatggaa cggggccccc agactctgaa
     2461 ggaaacatct tttaatcagg cctatggtcg tgatttaatg gaggcccaag agtggtgcag
     2521 gaagtacatg aaatcaggga atgtcaagga cctcacccaa gcctgggacc tctattatca
     2581 tgtgttccga cgaatctcaa agcagtctgg atccggatca ggctccggag acgggtcaag
     2641 cgtctctgga tctggagagg gaaggggaag cctcctaact tgcggagatg tcgaggagaa
     2701 tcctggccca cctgccgcta agcgtgtcaa gttggactct agagaacgcc cgtatgcttg
     2761 ccctgtcgag tcctgcgatc gccgcttttc tcgctcggat gagcttaccc gccatatccg
     2821 catccacaca ggccagaagc ccttccagtg tcgaatctgc atgcgtaact tcagtcgtag
     2881 tgaccacctt accacccaca tccgcaccca cacaggcggc ggccgcagga ggaagaaacg
     2941 caccagcata gagactaaca tccgtgtggc cttagagaag agtttcttgg agaatcaaaa
     3001 gcctacctcg gaagagatca ctatgattgc tgatcagctc aatatggaaa aagaggtgat
     3061 tcgtgtttgg ttctgtaacc gccgccagaa agaaaaaaga atcaacaagg gcggcgtgca
     3121 ggtggagact atctccccag gcgacgggcg caccttcccc aagcgcggcc agacctgcgt
     3181 ggtgcactac accgggatgc ttgaagatgg aaagaaattt gattcctccc gggacagaaa
     3241 caagcccttt aagtttatgc taggcaagca ggaggtgatc cgaggctggg aagaaggggt
     3301 tgcccagatg agtgtgggtc agagagccaa actgactata tctccagatt atgcctatgg
     3361 tgccactggg cacccaggca tcatcccacc acatgccact ctcgtctttg atgtggagct
     3421 tctaaaactg gaataacata tgaaataaaa tacgaaatgc gtacgtaatg atgggcgcta
     3481 aattaatgat gggcggagtt gtaatgatgg gcgctcgggt aatgatgggc gcccgtctaa
     3541 tgatgggcga gaagttaatg atgggcgctc gtttaatgat gggcgtctag ctaatgatgg
     3601 gcgggcgatt aatgatgggc gaaggactaa tgatgggcgc agcggtaatg atgggcgatc
     3661 agctaatgat gggcgacgcg aattaattca acattttgac acccccataa tatttttcca
     3721 gaattaacag tataaattgc atctcttgtt caagagttcc ctatcactct ctttaatcac
     3781 tactcacagt aacctcaact cctgcgccgc caccatggtg agcaagggcg aggagctgtt
     3841 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     3901 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     3961 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcct
     4021 gatgtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     4081 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     4141 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     4201 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     4261 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     4321 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     4381 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgag
     4441 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     4501 gatcactctc ggcatggacg agctgtacaa gtaagcctaa taaagagctc agatgcatgc
     4561 atcagagtgt gttggttttt tgtgtgtcgg accgagcggc cgcaggaacc ccatgatcgg
     4621 attagca
//
