LOCUS       sequence-269633-        5390 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FRET Standard.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5390)
  AUTHORS   Reissaus CA, Day KH, Mirmira RG, Dunn KW, Pavalko FM, Day RN
  TITLE     PIE-FLIM Measurements of Two Different FRET-Based Biosensor 
            Activities in the Same Living Cells.
  JOURNAL   Biophys J. 2020 Apr 21;118(8):1820-1829. doi: 
            10.1016/j.bpj.2020.03.003. Epub 2020 Mar 10.
  PUBMED    32191861
REFERENCE   2  (bases 1 to 5390)
  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"; doi: 
            "10.1016/j.bpj.2020.03.003"; journalName: "Biophys J"; date: 
            "2020-04-21- 21"; volume: "118"; issue: "8"; pages: "1820-1829"
COMMENT     Sequence Label: Scarlet I-FRET 10-NeonGreen
FEATURES             Location/Qualifiers
     source          1..5390
                     /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             613..1308
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(746..764)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1014..1033
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      1339..1348
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1345..2055
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      1600..1609
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2178..2299
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2215..2234)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2269..2288
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2306..2761)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2443..2464)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2655..2674
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2788..2892
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2894..3251
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2894..3085
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2917..2937)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3102..3237
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3286..4080
                     /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             3286..4080
                     /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(3340..3359)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3950..3969
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4268..4287)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4312..4359
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4688..5276
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5177..5196
                     /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 gcagtgatca aggagttcat gcggttcaag
      661 gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc
      721 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc
      781 ttctcctggg acatcctgtc ccctcagttc atgtacggct ccagggcctt catcaagcac
      841 cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa gtgggagcgc
      901 gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc cctggaggac
      961 ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga cggccccgta
     1021 atgcagaaga agacaatggg ctgggaagca tccaccgagc ggttgtaccc cgaggacggc
     1081 gtgctgaagg gcgacattaa gatggccctg cgcctgaagg acggcggccg ctacctggcg
     1141 gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc ctacaacgtc
     1201 gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga acagtacgaa
     1261 cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc cggactcaga
     1321 tcttcggatc caccggtcgc caccatggtg agcaagggcg aggaggataa catggcctct
     1381 ctcccagcga cacatgagtt acacatcttt ggctccatca acggtgtgga ctttgacatg
     1441 gtgggtcagg gcaccggcaa tccaaatgat ggttatgagg agttaaacct gaagtccacc
     1501 aagggtgacc tccagttctc cccctggatt ctggtccctc atatcgggta tggcttccat
     1561 cagtacctgc cctaccctga cgggatgtcg cctttccagg ccgccatggt agatggctcc
     1621 ggctaccaag tccatcgcac aatgcagttt gaagatggtg cctcccttac tgttaactac
     1681 cgctacacct acgagggaag ccacatcaaa ggagaggccc aggtgaaggg gactggtttc
     1741 cctgctgacg gtcctgtgat gaccaactcg ctgaccgctg cggactggtg caggtcgaag
     1801 aagacttacc ccaacgacaa aaccatcatc agtaccttta agtggagtta caccactgga
     1861 aatggcaagc gctaccggag cactgcgcgg accacctaca cctttgccaa gccaatggcg
     1921 gctaactatc tgaagaacca gccgatgtac gtgttccgta agacggagct caagcactcc
     1981 aagaccgagc tcaacttcaa ggagtggcaa aaggccttta ccgatgtgat gggcatggac
     2041 gagctgtaca agtaaagcgg ccgcgactct agatcataat cagccatacc acatttgtag
     2101 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     2161 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2221 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     2281 aactcatcaa tgtatcttaa cgcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     2341 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     2401 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     2461 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     2521 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     2581 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     2641 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     2701 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     2761 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2821 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     2881 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     2941 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3001 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3061 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3121 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3181 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3241 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3301 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3361 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3421 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     3481 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3541 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3601 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3661 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3721 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     3781 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     3841 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     3901 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     3961 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4021 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4081 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4141 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4201 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     4261 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     4321 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     4381 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     4441 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     4501 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     4561 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4621 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4681 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4741 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4801 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     4861 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4921 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4981 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5041 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5101 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5161 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5221 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5281 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5341 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat
//
