LOCUS       sequence-292554-        5389 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 5389)
  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 5389)
  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..5389
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        63..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      609..618
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             615..1310
                     /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(748..766)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1016..1035
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      1341..1350
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1347..2057
                     /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      1602..1611
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2179..2300
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2216..2235)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2270..2289
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2307..2762)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2444..2465)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2656..2675
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2789..2893
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2895..3252
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2895..3086
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2918..2938)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3103..3238
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3287..4081
                     /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             3287..4081
                     /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(3341..3360)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3951..3970
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4269..4288)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4313..4360
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4689..5277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5178..5197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      601 taccggtcgc caccatggtg agcaagggcg aggcagtgat caaggagttc atgcggttca
      661 aggtgcacat ggagggctcc atgaacggcc acgagttcga gatcgagggc gagggcgagg
      721 gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc
      781 ccttctcctg ggacatcctg tcccctcagt tcatgtacgg ctccagggcc ttcatcaagc
      841 accccgccga catccccgac tactataagc agtccttccc cgagggcttc aagtgggagc
      901 gcgtgatgaa cttcgaggac ggcggcgccg tgaccgtgac ccaggacacc tccctggagg
      961 acggcaccct gatctacaag gtgaagctcc gcggcaccaa cttccctcct gacggccccg
     1021 taatgcagaa gaagacaatg ggctgggaag cgtccaccga gcggttgtac cccgaggacg
     1081 gcgtgctgaa gggcgacatt aagatggccc tgcgcctgaa ggacggcggc cgctacctgg
     1141 cggacttcaa gaccacctac aaggccaaga agcccgtgca gatgcccggc gcctacaacg
     1201 tcgaccgcaa gttggacatc acctcccaca acgaggacta caccgtggtg gaacagtacg
     1261 aacgctccga gggccgccac tccaccggcg gcatggacga gctgtacaag tccggactca
     1321 gatcttcgga tccaccggtc gccaccatgg tgagcaaggg cgaggaggat aacatggcct
     1381 ctctcccagc gacacatgag ttacacatct ttggctccat caacggtgtg gactttgaca
     1441 tggtgggtca gggcaccggc aatccaaatg atggttatga ggagttaaac ctgaagtcca
     1501 ccaagggtga cctccagttc tccccctgga ttctggtccc tcatatcggg tatggcttcc
     1561 atcagtacct gccctaccct gacgggatgt cgcctttcca ggccgccatg gtagatggct
     1621 ccggctacca agtccatcgc acaatgcagt ttgaagatgg tgcctccctt actgttaact
     1681 accgctacac ctacgaggga agccacatca aaggagaggc ccaggtgaag gggactggtt
     1741 tccctgctga cggtcctgtg atgaccaact cgctgaccgc tgcggactgg tgcaggtcga
     1801 agaagactta ccccaacgac aaaaccatca tcagtacctt taagtggagt tacaccactg
     1861 gaaatggcaa gcgctaccgg agcactgcgc ggaccaccta cacctttgcc aagccaatgg
     1921 cggctaacta tctgaagaac cagccgatgt acgtgttccg taagacggag ctcaagcact
     1981 ccaagaccga gctcaacttc aaggagtggc aaaaggcctt taccgatgtg atgggcatgg
     2041 acgagctgta caagtaagcg gccgcgactc tagatcataa tcagccatac cacatttgta
     2101 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     2161 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     2221 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     2281 aaactcatca atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc
     2341 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2401 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2461 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2521 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2581 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2641 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2701 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2761 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2821 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2881 aaaaaggaag agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt
     2941 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3001 gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     3061 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     3121 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     3181 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     3241 ggcttttgca aagatcgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga
     3301 tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc
     3361 acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc
     3421 ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc
     3481 gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac
     3541 tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc
     3601 tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac
     3661 gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg
     3721 tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct
     3781 cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt
     3841 cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg
     3901 attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac
     3961 ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg
     4021 tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg
     4081 agcgggactc tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat
     4141 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     4201 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg
     4261 aggctaactg aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata
     4321 aaaagacaga ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     4381 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     4441 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     4501 gtcggggcgg caggccctgc catagcctca ggttactcat atatacttta gattgattta
     4561 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     4621 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     4681 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     4741 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     4801 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     4861 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     4921 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     4981 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     5041 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     5101 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     5161 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     5221 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     5281 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     5341 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgc
//
