LOCUS       sequence-379347-        5671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of EGFP to mScarlet-I fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5671)
  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 5671)
  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-mScarletI-antiFRET-P2A-EGFP
FEATURES             Location/Qualifiers
     source          1..5671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(205..224)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    249..296
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      625..1213
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1114..1133
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1361..1664
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1666..1868
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1819..1839
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2071..2093
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2155..2174
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2216..2334
                     /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             2409..2426
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2436..2453
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2472..2481
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2478..3173
                     /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(2611..2629)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2879..2898
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3294..3350
                     /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      3369..3378
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3375..3377,3378..3380,3381..4094)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3420..3441)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3681..3700)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4028..4049
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4187..4206)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4252..4307
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4306..4325)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4395..4442
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4468..4572
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4597..4617)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4641..4851
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4663..4859
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4710..4845
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(4894..5671,1..17)
                     /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(4894..5671,1..17)
                     /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(4948..4967)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5558..5577
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
       61 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
      121 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
      181 cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg
      241 cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa
      301 acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg
      361 ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc
      421 agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat
      481 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
      541 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
      601 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
      661 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
      721 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
      781 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
      841 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
      901 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
      961 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     1021 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     1081 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     1141 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     1201 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     1261 ttttgctcac atgttctttc ctgcgttatc ccctgaggcc ggcctattaa tagtaatcaa
     1321 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     1381 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     1441 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     1501 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc cctattgacg
     1561 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta cgggactttc
     1621 ctacttggca gtacatctac gtattagtca tcgctattac catgctgatg cggttttggc
     1681 agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     1741 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     1801 ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     1861 gcagacgtcg tttagtgaac cgtcagatca ctagatgctt tattgcggta gtttatcaca
     1921 gttaaattgc taacgccagt ctcgaactta acgtgcagaa gttggtcgtg aggcactggg
     1981 caggtaagta tcgggccctt tgtgcggggg gagcggctcg gggctgtccg cggggggacg
     2041 gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct
     2101 ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg
     2161 tgctggttat tgtgctgtct catcattttg gcaaagaatt ggatcggacc gaaatcacca
     2221 caattcagca aattgtgaac atcatcacgt tcatctttcc ctggttgcca aaggcccatt
     2281 ttcctgtcag taacgagaag gtcgcgaatt caggcgcttt ttagactggt cgtagggaga
     2341 ccacaacggt ttccctctag aaataatttt gtttaactat aagaaggaga tataccatgc
     2401 ggggttctca tcatcaccac catcacagca gcggcctggt gccgcgcggc agctcgctag
     2461 cgctgccggt cgccaccatg gtgagcaagg gcgaggcagt gatcaaggag ttcatgcggt
     2521 tcaaggtgca catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg
     2581 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc
     2641 tgcccttctc ctgggacatc ctgtcccctc agttcatgta cggctccagg gccttcatca
     2701 agcaccccgc cgacatcccc gactactata agcagtcctt ccccgagggc ttcaagtggg
     2761 agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt gacccaggac acctccctgg
     2821 aggacggcac cctgatctac aaggtgaagc tccgcggcac caacttccct cctgacggcc
     2881 ccgtaatgca gaagaagaca atgggctggg aagcgtccac cgagcggttg taccccgagg
     2941 acggcgtgct gaagggcgac attaagatgg ccctgcgcct gaaggacggc ggccgctacc
     3001 tggcggactt caagaccacc tacaaggcca agaagcccgt gcagatgccc ggcgcctaca
     3061 acgtcgaccg caagttggac atcacctccc acaacgagga ctacaccgtg gtggaacagt
     3121 acgaacgctc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtcgacca
     3181 gatctctggc cgaggcggca gctaaggagg ccgcggcgaa ggaggcagcc gcgaaagaag
     3241 cagccgcaaa ggaagcggcc gccaaggctg ctgcaagatc tctcggatcg ggtgctacta
     3301 acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct tcgctagcgc
     3361 tggatcccgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     3421 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     3481 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     3541 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     3601 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     3661 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     3721 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     3781 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     3841 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     3901 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     3961 tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc
     4021 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     4081 agctgtacaa gtaaaagctt gtgattaacc tcaggtgcag gctgcctatc agaaggtggt
     4141 ggctggtgtg gccaatgccc tggctcacaa ataccactga gatcgatctt tttccctctg
     4201 ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga
     4261 aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat
     4321 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat
     4381 atgcccatat gtaactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     4441 tgctgaaagc atgctcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4501 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4561 aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg
     4621 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4681 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4741 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4801 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     4861 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     4921 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4981 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     5041 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     5101 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     5161 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     5221 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     5281 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     5341 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     5401 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     5461 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     5521 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     5581 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     5641 gattcgcagc gcatcgcctt ctatcgcctt c
//
