LOCUS       sequence-379345-        5671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of mTurquoise2_T203Y 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-mTurquoise2_T203Y
FEATURES             Location/Qualifiers
     source          1..5671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(209..228)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    253..300
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      629..1217
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1118..1137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1365..1668
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1670..1872
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1823..1843
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2075..2097
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2159..2178
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2220..2338
                     /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             2413..2430
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2440..2457
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2476..2485
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2482..3177
                     /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(2615..2633)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2883..2902
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3298..3354
                     /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      3373..3382
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3379..3381,3382..3384,3385..4098)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3424..3445)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3685..3704)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4032..4053
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4191..4210)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4256..4311
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4310..4329)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4399..4446
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4472..4576
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4601..4621)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4645..4855
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4667..4863
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4714..4849
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(4898..5671,1..21)
                     /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(4898..5671,1..21)
                     /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(4952..4971)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5562..5581
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
       61 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
      121 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
      181 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
      241 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc
      301 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
      361 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
      421 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat
      481 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
      541 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
      601 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
      661 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
      721 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
      781 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
      841 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
      901 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
      961 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     1021 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     1081 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     1141 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     1201 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     1261 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ggccggccta ttaatagtaa
     1321 tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg
     1381 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     1441 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     1501 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt
     1561 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac
     1621 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct gatgcggttt
     1681 tggcagtaca ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac
     1741 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     1801 cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     1861 ataagcagac gtcgtttagt gaaccgtcag atcactagat gctttattgc ggtagtttat
     1921 cacagttaaa ttgctaacgc cagtctcgaa cttaacgtgc agaagttggt cgtgaggcac
     1981 tgggcaggta agtatcgggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg
     2041 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     2101 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     2161 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcg gaccgaaatc
     2221 accacaattc agcaaattgt gaacatcatc acgttcatct ttccctggtt gccaaaggcc
     2281 cattttcctg tcagtaacga gaaggtcgcg aattcaggcg ctttttagac tggtcgtagg
     2341 gagaccacaa cggtttccct ctagaaataa ttttgtttaa ctataagaag gagatatacc
     2401 atgcggggtt ctcatcatca ccaccatcac agcagcggcc tggtgccgcg cggcagctcg
     2461 ctagcgctgc cggtcgccac catggtgagc aagggcgagg cagtgatcaa ggagttcatg
     2521 cggttcaagg tgcacatgga gggctccatg aacggccacg agttcgagat cgagggcgag
     2581 ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac caagggtggc
     2641 cccctgccct tctcctggga catcctgtcc cctcagttca tgtacggctc cagggccttc
     2701 atcaagcacc ccgccgacat ccccgactac tataagcagt ccttccccga gggcttcaag
     2761 tgggagcgcg tgatgaactt cgaggacggc ggcgccgtga ccgtgaccca ggacacctcc
     2821 ctggaggacg gcaccctgat ctacaaggtg aagctccgcg gcaccaactt ccctcctgac
     2881 ggccccgtaa tgcagaagaa gacaatgggc tgggaagcgt ccaccgagcg gttgtacccc
     2941 gaggacggcg tgctgaaggg cgacattaag atggccctgc gcctgaagga cggcggccgc
     3001 tacctggcgg acttcaagac cacctacaag gccaagaagc ccgtgcagat gcccggcgcc
     3061 tacaacgtcg accgcaagtt ggacatcacc tcccacaacg aggactacac cgtggtggaa
     3121 cagtacgaac gctccgaggg ccgccactcc accggcggca tggacgagct gtacaagtcg
     3181 accagatctc tggccgaggc ggcagctaag gaggccgcgg cgaaggaggc agccgcgaaa
     3241 gaagcagccg caaaggaagc ggccgccaag gctgctgcaa gatctctcgg atcgggtgct
     3301 actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg accttcgcta
     3361 gcgctggatc ccgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
     3421 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
     3481 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
     3541 cccgtgccct ggcccaccct cgtgaccacc ctgtcctggg gcgtgcagtg cttcgcccgc
     3601 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
     3661 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
     3721 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
     3781 ggcaacatcc tggggcacaa gctggagtac aactacttta gcgacaacgt ctatatcacc
     3841 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
     3901 ggcggcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     3961 ctgctgcccg acaaccacta cctgagctac cagtccaagc tgagcaaaga ccccaacgag
     4021 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     4081 gacgagctgt acaagtaaaa gcttgtgatt aacctcaggt gcaggctgcc tatcagaagg
     4141 tggtggctgg tgtggccaat gccctggctc acaaatacca ctgagatcga tctttttccc
     4201 tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa
     4261 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg
     4321 acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca
     4381 acatatgccc atatgtaact agcataaccc cttggggcct ctaaacgggt cttgaggggt
     4441 tttttgctga aagcatgctc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     4501 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     4561 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     4621 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     4681 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4741 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4801 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4861 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4921 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4981 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     5041 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     5101 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     5161 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     5221 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     5281 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     5341 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     5401 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     5461 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     5521 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     5581 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     5641 tcccgattcg cagcgcatcg ccttctatcg c
//
