LOCUS       sequence-377506-        5288 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 5288)
  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 5288)
  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: N1-G-Ca-FLITS
FEATURES             Location/Qualifiers
     source          1..5288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        59..362
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        363..566
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     517..537
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    589..669
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      671..680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             692..748
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             773..1021
                     /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(1085..1106)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1346..1365)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2074..2195
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2111..2130)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2165..2184
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2202..2657)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2339..2360)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2551..2570
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2684..2788
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2790..3147
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2790..2981
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2813..2833)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2998..3133
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3182..3976
                     /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             3182..3976
                     /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(3236..3255)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3846..3865
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4164..4183)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4208..4255
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4584..5172
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5073..5092
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
       61 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
      121 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
      181 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa
      241 gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca
      301 tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc gctattacca
      361 tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac tcacggggat
      421 ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
      481 actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt aggcgtgtac
      541 ggtgggaggt ctatataagc agagctggtt tagtgaaccg tcagatccgc tagcgctacc
      601 ggactcagat ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga
      661 tccaccggtc gccaccatgg tcgactcttc acgtcgtaag tggaataagg caggtcacgc
      721 agtcagagct ataggtcggc tgagctcacc cgtggctgtc tatatcaccg ccgacaagca
      781 gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg gcggcgtgca
      841 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
      901 caaccactac ctgagctacc agtccaagct gagcaaagac cccaacgaga agcgcgatca
      961 catggtccta ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     1021 ccagggtggc agcggtggca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     1081 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     1141 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1201 gcccgtgccc tggcccaccc tcgtgaccac cctgtcctgg ggcgtgcagt gcttcgcccg
     1261 ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt
     1321 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     1381 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     1441 cggcaacatc ctggggcaca agctggagta caactactat agcgacgata cgcgtgacca
     1501 actgactgaa gagcagatcg cagaatttaa agaggctttc tccctatttg acaaggacgg
     1561 ggatgggacg ataacaacca aggagctggg gacggtgatg cggtctctgg ggcagaaccc
     1621 cacagaagca gagctgcagg acatgatcaa tgaagtagat gccgacggtg acggcacatt
     1681 cgacttccct gagttcctga cgatgatggc aagaaaaatg aatgacacag acagtgaaga
     1741 ggaaattaga gaagcgttcc gcgtgtttga taaggacggc aatggctaca tcggcgcagc
     1801 agagcttcgc cacgtgatga cagaccttgg agagaagtta acagatgagg aggttgatga
     1861 aatgatcagg gtagcagaca tcgatgggga tggtcaggta aactacgaag agtttgtcca
     1921 aatgatgaca gcgaagtagg tgtacaagta aagcggccgc gactctagat cataatcagc
     1981 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     2041 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     2101 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2161 agttgtggtt tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat
     2221 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     2281 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2341 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2401 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2461 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2521 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2581 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2641 gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     2701 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     2761 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     2821 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2881 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     2941 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     3001 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     3061 atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc
     3121 ttttttggag gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg
     3181 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3241 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3301 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3361 gcaagacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3421 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3481 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3541 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3601 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3661 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga
     3721 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3781 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3841 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3901 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     3961 tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac
     4021 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     4081 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     4141 gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc
     4201 gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa
     4261 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     4321 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     4381 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata
     4441 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     4501 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     4561 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     4621 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     4681 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg
     4741 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     4801 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     4861 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     4921 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     4981 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     5041 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     5101 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     5161 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     5221 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     5281 atgcatta
//
