LOCUS       sequence-218708-        5421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Localization: Cytosolic FRET, Excitation: , Emission: .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5421)
  AUTHORS   Davidson Lab
  TITLE     FRET Indicators - Live and Fixed Cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5421)
  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: 
            "Unpublished"
COMMENT     Sequence Label: mTagBFP-mEGFP-FRET
FEATURES             Location/Qualifiers
     source          1..5421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        419..622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     573..593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             692..1390
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     regulatory      1415..1424
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1421..1423,1424..1426,1427..2140)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1466..1487)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1727..1746)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2074..2095
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2263..2384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2300..2319)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2354..2373
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2391..2846)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2528..2549)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2740..2759
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2873..2977
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2979..3336
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2979..3170
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3002..3022)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3187..3322
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3371..4165
                     /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             3371..4165
                     /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(3425..3444)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4035..4054
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4353..4372)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4397..4444
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4773..5361
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5262..5281
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat gcattagtta
       61 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      121 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      181 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      241 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      301 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      361 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      421 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      481 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      541 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      601 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc gctaccggac
      661 tcagatctcg agctcaagct tcgaattcac catgagcgag ctgattaagg agaacatgca
      721 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg
      781 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
      841 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat
      901 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg
      961 ggagagagtc accacatacg aagacggggg cgtgctgacc gctacccagg acaccagcct
     1021 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg
     1081 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc tgtaccccgc
     1141 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct
     1201 gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc
     1261 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgagaccta
     1321 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca
     1381 caagcttaat tccggactca gatccccacc ggtcgccacc atggtgagca agggcgagga
     1441 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     1501 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     1561 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     1621 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1681 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     1741 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1801 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     1861 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     1921 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1981 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa
     2041 gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2101 cgccgggatc actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc
     2161 ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
     2221 cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
     2281 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2341 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag
     2401 cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca
     2461 ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag
     2521 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
     2581 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
     2641 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
     2701 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
     2761 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
     2821 gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac
     2881 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     2941 ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg
     3001 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     3061 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca
     3121 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact
     3181 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     3241 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     3301 tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg
     3361 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     3421 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     3481 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     3541 gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     3601 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     3661 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     3721 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     3781 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     3841 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag
     3901 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     3961 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4021 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4081 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     4141 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     4201 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     4261 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     4321 gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa
     4381 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt
     4441 gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc
     4501 cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt
     4561 gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac
     4621 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4681 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4741 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4801 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4861 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     4921 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4981 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     5041 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     5101 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     5161 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     5221 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     5281 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5341 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5401 tgctggcctt ttgctcacat g
//
