LOCUS       sequence-9486-ex        5439 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 5439)
  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 5439)
  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: Lyn-FKBP-FKBP-CFP (LF2C)
FEATURES             Location/Qualifiers
     source          1..5439
                     /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      635..644
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             683..1003
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1010..1330
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /note="synthetic gene with alternative codons"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1010..1330
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1337..1363
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1379..1388
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1385..1387,1388..1390,1391..2104)
                     /codon_start=1
                     /product="enhanced CFP"
                     /label=ECFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1430..1451)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1691..1710)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2038..2059
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2227..2348
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2264..2283)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2318..2337
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2355..2810)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2492..2513)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2704..2723
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2837..2941
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2943..3300
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2943..3134
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2966..2986)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3151..3286
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3335..4129
                     /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             3335..4129
                     /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(3389..3408)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3999..4018
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4317..4336)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4361..4408
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4737..5325
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5226..5245
                     /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 atctcgagct caagcttcga attcgccacc atgggatgta taaaatcaaa
      661 agggaaagac agcgcgggag caggagtgca ggtggaaacc atctccccag gagacgggcg
      721 caccttcccc aagcgcggcc agacctgcgt ggtgcactac accgggatgc ttgaagatgg
      781 aaagaaattt gattcctccc gggacagaaa caagcccttt aagtttatgc taggcaagca
      841 ggaggtgatc cgaggctggg aagaaggggt tgcccagatg agtgtgggtc agagagccaa
      901 actgactata tctccagatt atgcctatgg tgccactggg cacccaggca tcatcccacc
      961 acatgccact ctcgtcttcg atgtggagct tctaaaactg gaaactagag gcgtgcaggt
     1021 cgagaccatc agccccggcg acggccgcac ctttcccaag agaggccaga cttgcgtggt
     1081 ccactacacc ggcatgctgg aggacggcaa gaagttcgac agcagccgcg accgcaacaa
     1141 gcccttcaag ttcatgctgg gcaaacagga agtgatccgc ggctgggagg aaggcgtggc
     1201 tcagatgagc gtggggcagc gggccaagct gaccatcagc cccgactatg cctacggcgc
     1261 caccggccac cccggcatca tcccccccca cgccaccctg gtgttcgacg tggagctgct
     1321 gaagctggag actagttatc cgtacgacgt accagactac gcaaaggatc caccggtcgc
     1381 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     1441 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     1501 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     1561 caccctcgtg accaccctga cctggggcgt gcagtgcttc agccgctacc ccgaccacat
     1621 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     1681 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     1741 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     1801 gcacaagctg gagtacaact acatcagcca caacgtctat atcaccgccg acaagcagaa
     1861 gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     1921 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1981 ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat
     2041 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     2101 gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt
     2161 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2221 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2281 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2341 gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     2401 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2461 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2521 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2581 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     2641 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     2701 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     2761 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     2821 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2881 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2941 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3001 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3061 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3121 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3181 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3241 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3301 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3361 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3421 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3481 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     3541 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3601 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3661 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3721 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3781 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3841 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     3901 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     3961 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     4021 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     4081 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4141 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4201 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4261 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     4321 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     4381 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     4441 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     4501 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     4561 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     4621 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4681 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4741 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4801 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4861 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4921 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4981 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     5041 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     5101 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5161 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5221 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5281 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5341 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5401 tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
