LOCUS       sequence-315356-        4646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FRB-greenNFAST in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4646)
  AUTHORS   Tebo AG, Moeyaert B, Thauvin M, Carlon-Andres I, Boken D, Volovitch 
            M, Padilla-Parra S, Dedecker P, Vriz S, Gautier A
  TITLE     Orthogonal fluorescent chemogenetic reporters for multicolor 
            imaging.
  JOURNAL   Nat Chem Biol. 2020 Aug 10. pii: 10.1038/s41589-020-0611-0. doi: 
            10.1038/s41589-020-0611-0.
  PUBMED    32778846
REFERENCE   2  (bases 1 to 4646)
  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 Chem 
            Biol. 2020 Aug 10. pii: 10.1038/s41589-020-0611-0. doi: 
            10.1038/s41589-020-0611-0."
COMMENT     Sequence Label: pAG460-FRB-greenNFAST
FEATURES             Location/Qualifiers
     source          1..4646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      555..564
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             564..593
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             600..869
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     polyA_signal    1374..1495
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1411..1430)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1465..1484
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1502..1957)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1639..1660)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1851..1870
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1984..2088
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2090..2447
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2090..2281
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2113..2133)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2298..2433
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2482..3276
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2482..3276
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2536..2555)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3146..3165
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3464..3483)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3508..3555
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3884..4472
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4373..4392
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggactcag atctgccacc atggaacaaa agcttatttc tgaagaggac ttggaattcg
      601 agatgtggca tgaaggcctg gaagaggcat ctcgtttgta ctttggggaa aggaacgtga
      661 aaggcatgtt tgaggtgctg gagcccttgc atgctatgat ggaacggggc ccccagactc
      721 tgaaggaaac atcctttaat caggcctatg gtcgagattt aatggaggcc caagagtggt
      781 gcaggaagta catgaaatca gggaatgtca aggacctcac ccaagcctgg gacctctatt
      841 atcatgtgtt ccgacgaatc tcaaagcagg tctccggagg aggcggcagc ggcggagggg
      901 gatccatgga gcatgttgcc tttggcagtg aggacatcga gaacactctg gccaaaatgg
      961 acgacgaaca actggatggg ttggcctttg gcgcaattca gctcgatggt gacgggaata
     1021 tcctgcagta caatgctgct gaaggagaca tcacaggcag agatcccaaa caggtgattg
     1081 ggaagaactt cttcaaggat gttgcaactg gaacggattc tcccgagttt taccgcaaat
     1141 tcaaggaagg cgtagcgtca gggaatctga acaccatgtt cgaatggatg ataccgacaa
     1201 gcaggggacc aaccaaggtc aaggtgcaca tgaagaaagc cctttcctaa tagcggccgc
     1261 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     1321 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     1381 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     1441 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg
     1501 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     1561 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     1621 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     1681 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     1741 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     1801 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     1861 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     1921 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     1981 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2041 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     2101 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2161 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2221 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2281 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2341 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     2401 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     2461 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     2521 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     2581 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     2641 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     2701 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     2761 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     2821 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     2881 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     2941 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3001 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3061 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3121 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3181 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3241 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     3301 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     3361 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3421 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
     3481 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
     3541 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     3601 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     3661 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     3721 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     3781 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3841 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3901 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3961 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4021 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4081 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4141 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4201 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4261 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     4321 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     4381 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     4441 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     4501 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     4561 tggataaccg tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta
     4621 gttcatagcc catatatgga gttccg
//
