LOCUS       sequence-377502-        5689 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded calcium sensor (GECI) G-Ca-FLITS that reports 
            with a lifetime change.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5689)
  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 5689)
  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: 4xmts-G-Ca-FLITS
FEATURES             Location/Qualifiers
     source          1..5689
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        66..369
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        370..573
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     524..544
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     618..637
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        618..636
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             650..724
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             746..820
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             842..916
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             938..1012
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     regulatory      1079..1088
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1100..1156
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             1181..1429
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELY"
     primer_bind     complement(1493..1514)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1754..1773)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2482..2603
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2519..2538)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2573..2592
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2610..3065)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2747..2768)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2959..2978
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3092..3196
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3198..3555
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3198..3389
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3221..3241)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3406..3541
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3590..4384
                     /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             3590..4384
                     /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(3644..3663)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4254..4273
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4572..4591)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4616..4663
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4992..5580
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5481..5500
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgcattagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
       61 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      121 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      181 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      241 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      301 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      361 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      421 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      481 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      541 cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct
      601 tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctta tgtccgtcct
      661 gacgccgctg ctgctgcggg gcttgacagg ctcggcccgg cggctcccag tgccgcgcgc
      721 caagatccat tcgttggggg atcccatgtc cgtcctgacg ccgctgctgc tgcggggctt
      781 gacaggctcg gcccggcggc tcccagtgcc gcgcgccaag atccattcgt tggggaagct
      841 tatgtccgtc ctgacgccgc tgctgctgcg gggcttgaca ggctcggccc ggcggctccc
      901 agtgccgcgc gccaagatcc attcgttggg ggatcccatg tccgtcctga cgccgctgct
      961 gctgcggggc ttgacaggct cggcccggcg gctcccagtg ccgcgcgcca agatccattc
     1021 gttggggaag cttcgaattc tgcagtcgac ggtaccgcgg gcccgggatc caccggtcgc
     1081 caccatggtc gactcttcac gtcgtaagtg gaataaggca ggtcacgcag tcagagctat
     1141 aggtcggctg agctcacccg tggctgtcta tatcaccgcc gacaagcaga agaacggcat
     1201 caaggccaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc tcgccgacca
     1261 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1321 gagctaccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca tggtcctact
     1381 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtacc agggtggcag
     1441 cggtggcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     1501 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     1561 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     1621 gcccaccctc gtgaccaccc tgtcctgggg cgtgcagtgc ttcgcccgct accccgacca
     1681 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     1741 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     1801 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     1861 ggggcacaag ctggagtaca actactatag cgacgatacg cgtgaccaac tgactgaaga
     1921 gcagatcgca gaatttaaag aggctttctc cctatttgac aaggacgggg atgggacgat
     1981 aacaaccaag gagctgggga cggtgatgcg gtctctgggg cagaacccca cagaagcaga
     2041 gctgcaggac atgatcaatg aagtagatgc cgacggtgac ggcacattcg acttccctga
     2101 gttcctgacg atgatggcaa gaaaaatgaa tgacacagac agtgaagagg aaattagaga
     2161 agcgttccgc gtgtttgata aggacggcaa tggctacatc ggcgcagcag agcttcgcca
     2221 cgtgatgaca gaccttggag agaagttaac agatgaggag gttgatgaaa tgatcagggt
     2281 agcagacatc gatggggatg gtcaggtaaa ctacgaagag tttgtccaaa tgatgacagc
     2341 gaagtaggtg tacaagtaaa gcggccgcga ctctagatca taatcagcca taccacattt
     2401 gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct gaaacataaa
     2461 atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta caaataaagc
     2521 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     2581 tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt
     2641 cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat
     2701 cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa
     2761 gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg
     2821 cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa
     2881 agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc
     2941 gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag
     3001 tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg
     3061 cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3121 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3181 ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg
     3241 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3301 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3361 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3421 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3481 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3541 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3601 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3661 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     3721 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     3781 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     3841 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     3901 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     3961 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4021 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     4081 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     4141 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4201 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4261 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4321 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4381 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4441 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4501 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccctagg
     4561 gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca
     4621 ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg cggggttcgg
     4681 tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca atacgcccgc
     4741 gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg gctcgcagcc
     4801 aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact ttagattgat
     4861 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4921 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4981 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     5041 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5101 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     5161 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5221 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5281 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5341 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     5401 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5461 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5521 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5581 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5641 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcca
//
