LOCUS       sequence-250349-        4647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FRB-NFAST in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4647)
  AUTHORS   Tebo AG, Gautier A
  TITLE     A split fluorescent reporter with rapid and reversible 
            complementation.
  JOURNAL   Nat Commun. 2019 Jun 27;10(1):2822. doi: 10.1038/s41467-019-10855-0.
  PUBMED    31249300
REFERENCE   2  (bases 1 to 4647)
  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 
            Commun."; date: "2019-06-27"; volume: "10(1):2822. doi"; pages: " 
            10.1038/s41467-019-10855-0"
COMMENT     Sequence Label: pAG148 FRB-NFAST
FEATURES             Location/Qualifiers
     source          1..4647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      615..624
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             624..653
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             660..929
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     polyA_signal    1435..1556
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1472..1491)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1526..1545
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1563..2018)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1700..1721)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1912..1931
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2045..2149
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2151..2508
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2151..2342
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2174..2194)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2359..2494
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2543..3337
                     /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             2543..3337
                     /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(2597..2616)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3207..3226
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3525..3544)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3569..3616
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3945..4533
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4434..4453
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctgccacc atggaacaaa agcttatttc tgaagaggac ttggaattcg
      661 agatgtggca tgaaggcctg gaagaggcat ctcgtttgta ctttggggaa aggaacgtga
      721 aaggcatgtt tgaggtgctg gagcccttgc atgctatgat ggaacggggc ccccagactc
      781 tgaaggaaac atcctttaat caggcctatg gtcgagattt aatggaggcc caagagtggt
      841 gcaggaagta catgaaatca gggaatgtca aggacctcac ccaagcctgg gacctctatt
      901 atcatgtgtt ccgacgaatc tcaaagcagg tctccggagg aggcggcagc ggcggagggg
      961 gatccatgga gcatgttgcc tttggcagtg aggacatcga gaacactctg gccaaaatgg
     1021 acgacggaca actggatggg ttggcctttg gcgcaattca gctcgatggt gacgggaata
     1081 tcctgcagta caatgctgct gaaggagaca tcacaggcag agatcccaaa caggtgattg
     1141 ggaagaactt cttcaaggat gttgcacctg gaacggattc tcccgagttt tacggcaaat
     1201 tcaaggaagg cgtagcgtca gggaatctga acaccatgtt cgaatggatg ataccgacaa
     1261 gcaggggacc aaccaaggtc aaggtgcaca tgaagaaagc cctttcctaa taggcggccg
     1321 cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     1381 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     1441 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     1501 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     1561 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     1621 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     1681 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     1741 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     1801 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     1861 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     1921 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     1981 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     2041 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2101 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     2161 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     2221 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     2281 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     2341 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     2401 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     2461 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     2521 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     2581 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     2641 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     2701 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     2761 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     2821 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     2881 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     2941 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     3001 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     3061 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     3121 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     3181 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     3241 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     3301 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     3361 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     3421 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     3481 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     3541 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     3601 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     3661 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     3721 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     3781 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3841 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3901 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3961 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4021 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4081 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4141 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4201 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4261 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4321 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4381 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4441 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4501 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4561 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4621 gtggataacc gtattaccgc catgcat
//
