LOCUS       sequence-377505-        5363 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 5363)
  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 5363)
  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: ER-G-Ca-FLITS-KDEL
FEATURES             Location/Qualifiers
     source          1..5363
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        6..309
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        310..513
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     464..484
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     sig_peptide     586..639
                     /codon_start=1
                     /product="signal sequence of chicken lysozyme"
                     /label=lysozyme signal sequence
                     /translation="MRSLLILVLCFLPLAALG"
     regulatory      688..697
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             709..765
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             790..1038
                     /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(1102..1123)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1363..1382)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2096..2217
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2133..2152)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2187..2206
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2224..2679)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2361..2382)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2573..2592
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2706..2810
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2812..3169
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2812..3003
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2835..2855)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3020..3155
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3204..3998
                     /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             3204..3998
                     /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(3258..3277)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3868..3887
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4186..4205)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4230..4277
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4606..5194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5095..5114
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
       61 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      121 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      181 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      241 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      301 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      361 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      421 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      481 cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaaccgtca gatccgctag
      541 cgctaccgga ctcagatctc gagctcaagc ttcgaattcg ccaccatgag gtctttgcta
      601 atcttggtgc tttgcttcct gcccctggct gctctgggga aagtctttgg acgatgtgag
      661 ctggcagcgg ctatgaagcc accggtcgcc accatggtcg actcttcacg tcgtaagtgg
      721 aataaggcag gtcacgcagt cagagctata ggtcggctga gctcacccgt ggctgtctat
      781 atcaccgccg acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc
      841 gaggacggcg gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
      901 cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccaagctgag caaagacccc
      961 aacgagaagc gcgatcacat ggtcctactg gagttcgtga ccgccgccgg gatcactctc
     1021 ggcatggacg agctgtacca gggtggcagc ggtggcatgg tgagcaaggg cgaggagctg
     1081 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1141 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     1201 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gtcctggggc
     1261 gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1321 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     1381 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     1441 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctactatagc
     1501 gacgatacgc gtgaccaact gactgaagag cagatcgcag aatttaaaga ggctttctcc
     1561 ctatttgaca aggacgggga tgggacgata acaaccaagg agctggggac ggtgatgcgg
     1621 tctctggggc agaaccccac agaagcagag ctgcaggaca tgatcaatga agtagatgcc
     1681 gacggtgacg gcacattcga cttccctgag ttcctgacga tgatggcaag aaaaatgaat
     1741 gacacagaca gtgaagagga aattagagaa gcgttccgcg tgtttgataa ggacggcaat
     1801 ggctacatcg gcgcagcaga gcttcgccac gtgatgacag accttggaga gaagttaaca
     1861 gatgaggagg ttgatgaaat gatcagggta gcagacatcg atggggatgg tcaggtaaac
     1921 tacgaagagt ttgtccaaat gatgacagcg aaggtgtaca aggatgaatt gtaagcggcc
     1981 gcgactctag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     2041 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     2101 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     2161 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg
     2221 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     2281 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     2341 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     2401 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     2461 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     2521 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     2581 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     2641 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
     2701 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2761 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg
     2821 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     2881 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     2941 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     3001 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3061 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     3121 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag
     3181 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     3241 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     3301 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     3361 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
     3421 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     3481 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     3541 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     3601 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     3661 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     3721 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     3781 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     3841 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     3901 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     3961 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     4021 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     4081 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4141 ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga
     4201 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     4261 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     4321 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     4381 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     4441 agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4501 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     4561 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     4621 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     4681 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     4741 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     4801 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     4861 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4921 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4981 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5041 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5101 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5161 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5221 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5281 tgtggataac cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat
     5341 tagttcatag cccatatatg gag
//
