LOCUS       sequence-377483-        5288 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded calcium sensor (GECI) Tq-Ca-FLITS that reports 
            with lifetime and intensity changes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5288)
  AUTHORS   van der Linden FH, Mahlandt EK, Arts JJG, Beumer J, Puschhof J, de 
            Man SMA, Chertkova AO, Ponsioen B, Clevers H, van Buul JD, Postma M,
            Gadella TWJ Jr, Goedhart J
  TITLE     A turquoise fluorescence lifetime-based biosensor for quantitative 
            imaging of intracellular calcium.
  JOURNAL   Nat Commun. 2021 Dec 9;12(1):7159. doi: 10.1038/s41467-021-27249-w.
  PUBMED    34887382
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: "Nat 
            Commun."; date: "2021-12-9"; volume: "12(1):7159. doi"; pages: " 
            10.1038/s41467-021-27249-w"
COMMENT     Sequence Label: N1-Tq-Ca-FLITS
FEATURES             Location/Qualifiers
     source          1..5288
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        79..382
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        383..586
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     537..557
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    609..689
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      691..700
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             712..768
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             793..1041
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELY"
     primer_bind     complement(1105..1126)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1366..1385)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2094..2215
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2131..2150)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2185..2204
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2222..2677)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2359..2380)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2571..2590
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2704..2808
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2810..3167
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2810..3001
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2833..2853)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3018..3153
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3202..3996
                     /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             3202..3996
                     /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(3256..3275)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3866..3885
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4184..4203)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4228..4275
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4604..5192
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5093..5112
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgtattaccg ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag
       61 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      121 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      181 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      241 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      301 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      361 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      421 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      481 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      541 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
      601 tcagatccgc tagcgctacc ggactcagat ctcgagctca agcttcgaat tctgcagtcg
      661 acggtaccgc gggcccggga tccaccggtc gccaccatgg tcgactcttc acgtcgtaag
      721 tggaataagg caggtcacgc agtcagagct ataggtcggc tgagctcacc cgtggttgtc
      781 tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac
      841 atcgaggacg gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
      901 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccaagct gagcaaagac
      961 cccaacgaga agcgcgatca catggtccta ctggagttcg tgaccgccgc cgggatcact
     1021 ctcggcatgg acgagctgta ccagggtggc agcggtggca tggtgagcaa gggcgaggag
     1081 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1141 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1201 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgtcctgg
     1261 ggcgtgcagt gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1321 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1381 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1441 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactactat
     1501 agcgacaaca cgcgtgacca actgactgaa gagcagatcg cagaatttaa agaggctttc
     1561 tccctatttg acaaggacgg ggatgggacg ataacaacca aggagctggg gacggtgatg
     1621 cggtctctgg ggcagaaccc cacagaagca gagctgcagg acatgatcaa tgaagtagat
     1681 gccgacggtg acggcacatt cgacttccct gagttcctga cgatgatggc aagaaaaatg
     1741 aatgacacag acagtgaaga ggaaattaga gaagcgttcc gcgtgtttga taaggacggc
     1801 aatggctaca tcggcgcagc agagcttcgc cacgtgatga cagaccttgg agagaagtta
     1861 acagatgagg aggttgatga aatgatcagg gtagcagaca tcgatgggga tggtcaggta
     1921 aactacgaag agtttgtcca aatgatgaca gcgaagtagg tgtacaagta aagcggccgc
     1981 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     2041 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     2101 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     2161 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg
     2221 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     2281 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     2341 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     2401 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     2461 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     2521 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     2581 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     2641 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     2701 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2761 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     2821 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2881 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2941 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3001 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3061 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     3121 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     3181 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3241 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3301 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3361 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     3421 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3481 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3541 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3601 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3661 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3721 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3781 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3841 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3901 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3961 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4021 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4081 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4141 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
     4201 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
     4261 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     4321 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     4381 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     4441 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     4501 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     4561 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     4621 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     4681 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     4741 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     4801 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     4861 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     4921 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     4981 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     5041 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     5101 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     5161 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     5221 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     5281 tggataac
//
