LOCUS       sequence-334723-        5758 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses tandem mClover2-mRuby2 FRET pair with a 
            crowdedness-sensitve linker domain to sense cytosolic crowdedness..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5758)
  AUTHORS   Chen K, Yan R, Xiang L, Xu K
  TITLE     Excitation spectral microscopy for highly multiplexed fluorescence 
            imaging and quantitative biosensing.
  JOURNAL   Light Sci Appl. 2021 May 8;10(1):97. doi: 
            10.1038/s41377-021-00536-3.
  PUBMED    33963178
REFERENCE   2  (bases 1 to 5758)
  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: "Light Sci 
            Appl."; date: "2021-05-8"; volume: "10(1):97. doi"; pages: " 
            10.1038/s41377-021-00536-3"
COMMENT     Sequence Label: pmRuby2-CrowdingLinker-Clover
FEATURES             Location/Qualifiers
     source          1..5758
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      535..544
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             541..1251
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     regulatory      1639..1648
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1645..2364
                     /codon_start=1
                     /product="monomeric derivative of the bright green-yellow 
                     fluorescent protein Clover"
                     /label=mClover2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1690..1711)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2298..2319
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2486..2607
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2523..2542)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2577..2596
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2614..3069)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2751..2772)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2963..2982
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3096..3200
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3202..3559
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3202..3393
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3225..3245)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3410..3545
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3594..4388
                     /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             3594..4388
                     /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(3648..3667)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4258..4277
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4576..4595)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4620..4667
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4996..5584
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5485..5504
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagccacc
      541 atggtgtcta agggcgaaga gctgatcaag gaaaatatgc gtatgaaggt ggtcatggaa
      601 ggttcggtca acggccacca attcaaatgc acaggtgaag gagaaggcaa tccgtacatg
      661 ggaactcaaa ccatgaggat caaagtcatc gagggaggac ccctgccatt tgcctttgac
      721 attcttgcca cgtcgttcat gtatggcagc cgtactttta tcaagtaccc gaaaggcatt
      781 cctgatttct ttaaacagtc ctttcctgag ggttttactt gggaaagagt tacgagatac
      841 gaagatggtg gagtcgtcac cgtcatgcag gacaccagcc ttgaggatgg ctgtctcgtt
      901 taccacgtcc aagtcagagg ggtaaacttt ccctccaatg gtcccgtgat gcagaagaag
      961 accaagggtt gggagcctaa tacagagatg atgtatccag cagatggtgg tctgagggga
     1021 tacactcata tggcactgaa agttgatggt ggtggccatc tgtcttgctc tttcgtaaca
     1081 acttacaggt caaaaaagac cgtcgggaac atcaagatgc ccggtatcca tgccgttgat
     1141 caccgcctgg aaaggttaga ggaaagtgac aatgaaatgt tcgtagtaca acgcgaacac
     1201 gcagttgcca agttcgccgg gcttggtggt gggatggacg agctgtacaa gtccggactc
     1261 agatccccac cggtcggatc tggtggttcc ggcggttcag gtggatcggg aggaagtgga
     1321 ggtagcggtg ctgaagctgc agcaaaagag gcagctgcta aagaagctgc agctaaagaa
     1381 gcagctgcaa aagaagctgc tgcaaaggaa gctgcggcaa aggctggtag cggaggaagt
     1441 ggtggatcgg gaggttcagg aggttccgga ggatctggtg ctgaggctgc agctaaggaa
     1501 gcagcagcta aagaagcggc cgcaaaagag gctgcggcta aagaggcggc agccaaagag
     1561 gccgctgcca aggcaggttc gggcgggtct ggtggctcag gaggcagtgg cggaagcgga
     1621 ggctccgggc caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg
     1681 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtccgcggc
     1741 gagggcgagg gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc
     1801 aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcgt ggcctgcttc
     1861 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1921 tacgtccagg agcgcaccat ctctttcaag gacgacggta cctacaagac ccgcgccgag
     1981 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2041 gaggacggca acatcctggg gcacaagctg gagtacaact tcaacagcca caacgtctat
     2101 atcacggccg acaagcagaa gaacggcatc aaggctaact tcaagatccg ccacaacgtt
     2161 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     2221 cccgtgctgc tgcccgacaa ccactacctg agccatcagt ccgccctgag caaagacccc
     2281 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     2341 ggcatggacg agctgtacaa gtaagcggcc gcgactctag atcataatca gccataccac
     2401 atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca
     2461 taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata
     2521 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     2581 tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa
     2641 aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca
     2701 aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga
     2761 acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc
     2821 agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc
     2881 gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc
     2941 cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg
     3001 caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac
     3061 agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     3121 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     3181 aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt
     3241 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3301 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3361 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3421 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     3481 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     3541 aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg
     3601 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3661 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3721 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg
     3781 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3841 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3901 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3961 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4021 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4081 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg
     4141 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4201 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4261 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4321 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4381 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4441 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4501 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4561 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac
     4621 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt
     4681 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     4741 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     4801 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat
     4861 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     4921 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     4981 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     5041 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     5101 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     5161 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     5221 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     5281 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     5341 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     5401 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     5461 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     5521 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     5581 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     5641 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg ccatgcatta
     5701 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccg
//
