LOCUS       sequence-379343-        5755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of mCherry to mTurquoise2 fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5755)
  AUTHORS   van der Linden FH, Thornquist SC, ter Beek RM, Huijts JY, Hink MA, 
            Gadella Jr. TWJ, Maimon G, Goedhart J.
  TITLE     A green lifetime biosensor for calcium that remains bright over its 
            full dynamic range.
  JOURNAL   bioRxiv 2024.11.05.622032; doi: 
            https://doi.org/10.1101/2024.11.05.622032
REFERENCE   2  (bases 1 to 5755)
  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: "bioRxiv 
            2024.11.05.622032; doi: https://doi.org/10.1101/2024.11.05.622032"
COMMENT     Sequence Label: pFR-mTq2-antiFRET-P2A-mCherry
FEATURES             Location/Qualifiers
     source          1..5755
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(196..215)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    240..287
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      616..1204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1105..1124
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1352..1655
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1657..1859
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1810..1830
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2062..2084
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2146..2165
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2207..2325
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             2400..2417
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2427..2444
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2463..2472
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2469..2471,2472..2474,2475..3185)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2514..2535)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2775..2794)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3122..3143
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3306..3362
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     regulatory      3381..3390
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3387..4097
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3532..3550)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3800..3819
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4190..4209)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4255..4310
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4309..4328)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4398..4445
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4471..4575
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4577..4934
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4577..4768
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4600..4620)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4785..4920
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(4969..5755,1..8)
                     /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             join(4969..5755,1..8)
                     /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(5023..5042)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5633..5652
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
       61 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
      121 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
      181 tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac
      241 ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt
      301 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
      361 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
      421 agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat
      481 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
      541 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
      601 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
      661 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
      721 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
      781 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
      841 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
      901 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
      961 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1021 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1081 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1141 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1201 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1261 catgttcttt cctgcgttat cccctgaggc cggcctatta atagtaatca attacggggt
     1321 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     1381 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     1441 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     1501 acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac gtcaatgacg
     1561 gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt cctacttggc
     1621 agtacatcta cgtattagtc atcgctatta ccatgctgat gcggttttgg cagtacacca
     1681 atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca
     1741 atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aataaccccg
     1801 ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagacgtc
     1861 gtttagtgaa ccgtcagatc actagatgct ttattgcggt agtttatcac agttaaattg
     1921 ctaacgccag tctcgaactt aacgtgcaga agttggtcgt gaggcactgg gcaggtaagt
     1981 atcgggccct ttgtgcgggg ggagcggctc ggggctgtcc gcggggggac ggctgccttc
     2041 gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc tctagagcct
     2101 ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac gtgctggtta
     2161 ttgtgctgtc tcatcatttt ggcaaagaat tggatcggac cgaaatcacc acaattcagc
     2221 aaattgtgaa catcatcacg ttcatctttc cctggttgcc aaaggcccat tttcctgtca
     2281 gtaacgagaa ggtcgcgaat tcaggcgctt tttagactgg tcgtagggag accacaacgg
     2341 tttccctcta gaaataattt tgtttaacta taagaaggag atataccatg cggggttctc
     2401 atcatcacca ccatcacagc agcggcctgg tgccgcgcgg cagctcgcta gcgctgccgg
     2461 tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     2521 agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg
     2581 ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     2641 ggcccaccct cgtgaccacc ctgtcctggg gcgtgcagtg cttcgcccgc taccccgacc
     2701 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     2761 ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg
     2821 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     2881 tggggcacaa gctggagtac aactacttta gcgacaacgt ctatatcacc gccgacaagc
     2941 agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac ggcggcgtgc
     3001 agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg
     3061 acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc
     3121 acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt
     3181 acaagtcgac cagatctctg gccgaggcgg cagctaagga ggccgcggcg aaggaggcag
     3241 ccgcgaaaga agcagccgca aaggaagcgg ccgccaaggc tgctgcaaga tctctcggat
     3301 cgggtgctac taacttcagc ctgctgaagc aggctggaga cgtggaggag aaccctggac
     3361 cttcgctagc gctggatccc gccaccatgg tgagcaaggg cgaggaggat aacatggcca
     3421 tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt
     3481 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     3541 aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt
     3601 acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct
     3661 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg
     3721 tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca
     3781 ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct
     3841 ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc
     3901 tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg
     3961 tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg
     4021 actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg
     4081 acgagctgta caagtaaaag cttgtgatta acctcaggtg caggctgcct atcagaaggt
     4141 ggtggctggt gtggccaatg ccctggctca caaataccac tgagatcgat ctttttccct
     4201 ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa
     4261 ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga
     4321 catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa
     4381 catatgccca tatgtaacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     4441 ttttgctgaa agcatgctcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4501 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4561 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     4621 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     4681 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     4741 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     4801 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     4861 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     4921 taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa
     4981 gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg
     5041 gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc
     5101 ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca
     5161 gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc
     5221 actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca
     5281 tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat
     5341 acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca
     5401 cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg
     5461 ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc
     5521 gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct
     5581 ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct
     5641 acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac
     5701 ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagt
//
