LOCUS       sequence-377503-        5348 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 5348)
  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 5348)
  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: 3xnls-G-Ca-FLITS
FEATURES             Location/Qualifiers
     source          1..5348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        63..366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        367..570
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     521..541
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             654..674
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             678..698
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             702..722
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      735..744
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             756..812
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             837..1085
                     /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(1149..1170)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1410..1429)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2138..2259
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2175..2194)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2229..2248
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2266..2721)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2403..2424)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2615..2634
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2748..2852
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2854..3211
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2854..3045
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2877..2897)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3062..3197
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3246..4040
                     /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             3246..4040
                     /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(3300..3319)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3910..3929
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4228..4247)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4272..4319
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4648..5236
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5137..5156
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 attagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
       61 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      361 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      421 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      481 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      541 gtacggtggg aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcgc
      601 taccggactc agatctcgag ctcaagcttc gaattcccac catgggatca gatccaaaaa
      661 agaagagaaa ggtagacccg aagaaaaagc gtaaggtcga tcccaaaaag aaacggaaag
      721 tgggatcacc ggtcgccacc atggtcgact cttcacgtcg taagtggaat aaggcaggtc
      781 acgcagtcag agctataggt cggctgagct cacccgtggc tgtctatatc accgccgaca
      841 agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag gacggcggcg
      901 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
      961 ccgacaacca ctacctgagc taccagtcca agctgagcaa agaccccaac gagaagcgcg
     1021 atcacatggt cctactggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1081 tgtaccaggg tggcagcggt ggcatggtga gcaagggcga ggagctgttc accggggtgg
     1141 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     1201 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     1261 agctgcccgt gccctggccc accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg
     1321 cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     1381 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     1441 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     1501 aggacggcaa catcctgggg cacaagctgg agtacaacta ctatagcgac gatacgcgtg
     1561 accaactgac tgaagagcag atcgcagaat ttaaagaggc tttctcccta tttgacaagg
     1621 acggggatgg gacgataaca accaaggagc tggggacggt gatgcggtct ctggggcaga
     1681 accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac ggtgacggca
     1741 cattcgactt ccctgagttc ctgacgatga tggcaagaaa aatgaatgac acagacagtg
     1801 aagaggaaat tagagaagcg ttccgcgtgt ttgataagga cggcaatggc tacatcggcg
     1861 cagcagagct tcgccacgtg atgacagacc ttggagagaa gttaacagat gaggaggttg
     1921 atgaaatgat cagggtagca gacatcgatg gggatggtca ggtaaactac gaagagtttg
     1981 tccaaatgat gacagcgaag taggtgtaca agtaaagcgg ccgcgactct agatcataat
     2041 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     2101 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     2161 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     2221 ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta
     2281 atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg
     2341 ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg
     2401 ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa
     2461 aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg
     2521 ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt
     2581 gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg
     2641 ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta
     2701 atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     2761 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     2821 aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa
     2881 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2941 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3001 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3061 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3121 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     3181 aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga tgaggatcgt
     3241 ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc
     3301 tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc
     3361 tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg
     3421 aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag
     3481 ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg
     3541 ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg
     3601 caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac
     3661 atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg
     3721 acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc
     3781 ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg
     3841 aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc
     3901 aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc
     3961 gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc
     4021 ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc aagcgacgcc
     4081 caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg
     4141 aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt
     4201 cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac cggaaggaac
     4261 ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt cgtttgttca
     4321 taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg
     4381 gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg
     4441 cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag gttactcata
     4501 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4561 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4621 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4681 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4741 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4801 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     4861 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     4921 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     4981 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5041 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5101 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5161 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5221 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5281 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     5341 cgccatgc
//
