LOCUS       sequence-269640-        5399 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 5399)
  AUTHORS   Day RN, Day KH, Pavalko FM
  TITLE     Direct visualization by FRET-FLIM of a putative mechanosome complex 
            involving Src, Pyk2 and MBD2 in living MLO-Y4 cells.
  JOURNAL   PLoS One. 2021 Dec 23;16(12):e0261660. doi: 
            10.1371/journal.pone.0261660. eCollection 2021.
  PUBMED    34941939
REFERENCE   2  (bases 1 to 5399)
  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: "PLoS One.";
            date: "2021-12-23"; volume: "16(12):e0261660. doi"; pages: " 
            10.1371/journal.pone.0261660. eCollection 2021"
COMMENT     Sequence Label: Scarlet I-FRET 10-Cerulean3
FEATURES             Location/Qualifiers
     source          1..5399
                     /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             join(1345..1347,1348..1350,1351..2064)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1390..1411)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1651..1670)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1998..2019
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2187..2308
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2224..2243)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2278..2297
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2315..2770)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2452..2473)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2664..2683
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2797..2901
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2903..3260
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2903..3094
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2926..2946)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3111..3246
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3295..4089
                     /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             3295..4089
                     /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(3349..3368)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3959..3978
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4277..4296)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4321..4368
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4697..5285
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5186..5205
                     /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 aggagctgtt caccggggtg
     1381 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     1441 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     1501 aagctgcccg tgccctggcc caccctcgtg accaccctga gctggggcgt gcagtgcttc
     1561 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1621 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     1681 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1741 gaggacggca acatcctggg gcacaagctg gagtacaacg ccatccacgg caacgtctat
     1801 atcaccgccg acaagcagaa gaacggcatc aaggccaact tcggcctcaa ctgcaacatc
     1861 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1921 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc
     1981 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     2041 ggcatggacg agctgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca
     2101 catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac
     2161 ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat
     2221 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     2281 gtttgtccaa actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta
     2341 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     2401 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     2461 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     2521 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     2581 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     2641 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     2701 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     2761 cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2821 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2881 taatattgaa aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt
     2941 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3001 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     3061 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     3121 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     3181 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     3241 gaggcctagg cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt
     3301 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     3361 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     3421 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac
     3481 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     3541 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     3601 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     3661 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     3721 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     3781 caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag
     3841 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     3901 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     3961 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     4021 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     4081 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     4141 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4201 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4261 ctagggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     4321 cggcaataaa aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg
     4381 ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg
     4441 cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg
     4501 cagccaacgt cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga
     4561 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4621 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4681 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4741 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4801 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     4861 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4921 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4981 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5041 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5101 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5161 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5221 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5281 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5341 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
