LOCUS       sequence-257672-        5016 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5016)
  AUTHORS   Inoue T, Heo WD, Grimley JS, Wandless TJ, Meyer T
  TITLE     An inducible translocation strategy to rapidly activate and inhibit 
            small GTPase signaling pathways.
  JOURNAL   Nat Methods. 2005 Jun . 2(6):415-8.
  PUBMED    15908919
REFERENCE   2  (bases 1 to 5016)
  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 
            Methods. 2005 Jun . 2(6):415-8."
COMMENT     Sequence Label: YFP-tagged FRB (YR)
FEATURES             Location/Qualifiers
     source          1..5016
                     /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      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    1330..1395
                     /label=MCS
                     /note="multiple cloning site"
     CDS             1408..1686
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     polyA_signal    1804..1925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1841..1860)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1895..1914
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1932..2387)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2069..2090)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2281..2300
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2414..2518
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2520..2877
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2520..2711
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2543..2563)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2728..2863
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2912..3706
                     /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             2912..3706
                     /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(2966..2985)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3576..3595
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3894..3913)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3938..3985
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4314..4902
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4803..4822
                     /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 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccttcggc tacggcctgc agtgcttcgc ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag ctaccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcgagct caagcttcga attctgcagt cgacggtacc
     1381 gcgggcccgg gatccaccgg atctagaatc ctctggcatg agatgtggca tgaaggcctg
     1441 gaagaggcat ctcgtttgta ctttggggaa aggaacgtga aaggcatgtt tgaggtgctg
     1501 gagcccttgc atgctatgat ggaacggggc ccccagactc tgaaggaaac atcctttaat
     1561 caggcctatg gtcgagattt aatggaggcc caagagtggt gcaggaagta catgaaatca
     1621 gggaatgtca aggacctcct ccaagcctgg gacctctatt atcatgtgtt ccgacgaatc
     1681 tcaaagacta gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct
     1741 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     1801 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     1861 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     1921 tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta
     1981 aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga
     2041 atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa
     2101 cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga
     2161 accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc
     2221 taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga
     2281 agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg
     2341 cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt
     2401 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     2461 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc
     2521 tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2581 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2641 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2701 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2761 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     2821 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat
     2881 cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg
     2941 ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg
     3001 ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa
     3061 gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct
     3121 ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga
     3181 ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc
     3241 cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac
     3301 ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc
     3361 cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact
     3421 gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga
     3481 tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg
     3541 ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga
     3601 agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga
     3661 ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg
     3721 ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     3781 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3841 cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca
     3901 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     3961 acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct
     4021 gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca
     4081 ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc
     4141 cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa
     4201 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4261 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4321 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4381 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4441 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     4501 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4561 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4621 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4681 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4741 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4801 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4861 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4921 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4981 cctgattctg tggataaccg tattaccgcc atgcat
//
