LOCUS       sequence-334722-        5994 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mRuby2 and mClover2 in the cytoplasm as negative control 
            for FRET..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5994)
  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 5994)
  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-IRES-Clover
FEATURES             Location/Qualifiers
     source          1..5994
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      231..240
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             237..947
                     /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"
     misc_feature    983..1550
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1000..1552
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1144..1161)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1371..1390
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      1571..1580
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1577..2296
                     /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(1622..1643)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2230..2251
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2418..2539
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2455..2474)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2509..2528
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2546..3001)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2683..2704)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2895..2914
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3028..3132
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3134..3491
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3134..3325
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3157..3177)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3342..3477
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3526..4320
                     /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             3526..4320
                     /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(3580..3599)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4190..4209
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4508..4527)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4552..4599
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4928..5516
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5417..5436
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5691..5994
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gccaccatgg
      241 tgtctaaggg cgaagagctg atcaaggaaa atatgcgtat gaaggtggtc atggaaggtt
      301 cggtcaacgg ccaccaattc aaatgcacag gtgaaggaga aggcaatccg tacatgggaa
      361 ctcaaaccat gaggatcaaa gtcatcgagg gaggacccct gccatttgcc tttgacattc
      421 ttgccacgtc gttcatgtat ggcagccgta cttttatcaa gtacccgaaa ggcattcctg
      481 atttctttaa acagtccttt cctgagggtt ttacttggga aagagttacg agatacgaag
      541 atggtggagt cgtcaccgtc atgcaggaca ccagccttga ggatggctgt ctcgtttacc
      601 acgtccaagt cagaggggta aactttccct ccaatggtcc cgtgatgcag aagaagacca
      661 agggttggga gcctaataca gagatgatgt atccagcaga tggtggtctg aggggataca
      721 ctcatatggc actgaaagtt gatggtggtg gccatctgtc ttgctctttc gtaacaactt
      781 acaggtcaaa aaagaccgtc gggaacatca agatgcccgg tatccatgcc gttgatcacc
      841 gcctggaaag gttagaggaa agtgacaatg aaatgttcgt agtacaacgc gaacacgcag
      901 ttgccaagtt cgccgggctt ggtggtggga tggacgagct gtacaagtcc ggactcagat
      961 ccccaccggt taattaacga attccgcccc ccccccctaa cgttactggc cgaagccgct
     1021 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
     1081 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
     1141 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
     1201 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
     1261 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
     1321 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
     1381 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
     1441 atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
     1501 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca
     1561 accaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     1621 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtccgcg gcgagggcga
     1681 gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1741 cgtgccctgg cccaccctcg tgaccacctt cggctacggc gtggcctgct tcagccgcta
     1801 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1861 ggagcgcacc atctctttca aggacgacgg tacctacaag acccgcgccg aggtgaagtt
     1921 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1981 caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacgtct atatcacggc
     2041 cgacaagcag aagaacggca tcaaggctaa cttcaagatc cgccacaacg ttgaggacgg
     2101 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     2161 gctgcccgac aaccactacc tgagccatca gtccgccctg agcaaagacc ccaacgagaa
     2221 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     2281 cgagctgtac aagtaagcgg ccgcgactct agatcataat cagccatacc acatttgtag
     2341 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     2401 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2461 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     2521 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     2581 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     2641 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     2701 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     2761 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     2821 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     2881 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     2941 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     3001 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3061 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     3121 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     3181 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3241 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     3301 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc
     3361 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     3421 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     3481 gcttttgcaa agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3541 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3601 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3661 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg
     3721 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3781 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3841 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3901 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3961 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4021 gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc
     4081 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4141 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4201 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4261 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4321 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4381 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4441 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
     4501 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     4561 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
     4621 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
     4681 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
     4741 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
     4801 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4861 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4921 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4981 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     5041 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     5101 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     5161 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     5221 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     5281 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     5341 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     5401 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     5461 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     5521 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     5581 ttcctgcgtt atcccctgat tctgtggata accgtattac cgccatgcat tagttattaa
     5641 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     5701 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     5761 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     5821 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     5881 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     5941 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catg
//
