LOCUS       sequence-260197-        5348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Genetically encoded calcium sensor (GECI) that reports with lifetime
            and intensity changes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5348)
  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 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: "Nat 
            Commun."; date: "2021-12-9"; volume: "12(1):7159. doi"; pages: " 
            10.1038/s41467-021-27249-w"
COMMENT     Sequence Label: 3xnls-cpmTq2-Calcium-lifetime-sensor
FEATURES             Location/Qualifiers
     source          1..5348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             652..672
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             676..696
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             700..720
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      733..742
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             754..810
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             835..1083
                     /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(1147..1168)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1408..1427)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2136..2257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2173..2192)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2227..2246
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2264..2719)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2401..2422)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2613..2632
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2746..2850
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2852..3209
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2852..3043
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2875..2895)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3060..3195
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3244..4038
                     /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             3244..4038
                     /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(3298..3317)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3908..3927
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4226..4245)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4270..4317
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4646..5234
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5135..5154
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagct caagcttcga attcccacca tgggatcaga tccaaaaaag
      661 aagagaaagg tagacccgaa gaaaaagcgt aaggtcgatc ccaaaaagaa acggaaagtg
      721 ggatcaccgg tcgccaccat ggtcgactct tcacgtcgta agtggaataa ggcaggtcac
      781 gcagtcagag ctataggtcg gctgagctca cccgtggttg tctatatcac cgccgacaag
      841 cagaagaacg gcatcaaggc caacttcaag atccgccaca acatcgagga cggcggcgtg
      901 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
      961 gacaaccact acctgagcac ccagtccaag ctgagcaaag accccaacga gaagcgcgat
     1021 cacatggtcc tactggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     1081 taccagggtg gcagcggtgg catggtgagc aagggcgagg agctgttcac cggggtggtg
     1141 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     1201 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     1261 ctgcccgtgc cctggcccac cctcgtgacc accctgtcct ggggcgtgca gtgcttcgcc
     1321 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     1381 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     1441 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1501 gacggcaaca tcctggggca caagctggag tacaactact atagcgacaa cacgcgtgac
     1561 caactgactg aagagcagat cgcagaattt aaagaggctt tctccctatt tgacaaggac
     1621 ggggatggga cgataacaac caaggagctg gggacggtga tgcggtctct ggggcagaac
     1681 cccacagaag cagagctgca ggacatgatc aatgaagtag atgccgacgg tgacggcaca
     1741 ttcgacttcc ctgagttcct gacgatgatg gcaagaaaaa tgaatgacac agacagtgaa
     1801 gaggaaatta gagaagcgtt ccgcgtgttt gataaggacg gcaatggcta catcggcgca
     1861 gcagagcttc gccacgtgat gacagacctt ggagagaagt taacagatga ggaggttgat
     1921 gaaatgatca gggtagcaga catcgatggg gatggtcagg taaactacga agagtttgtc
     1981 caaatgatga cagcgaagta ggtgtacaag taaagcggcc gcgactctag atcataatca
     2041 gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga
     2101 acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg
     2161 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     2221 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat
     2281 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     2341 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt
     2401 ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa
     2461 accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg
     2521 tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga
     2581 cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct
     2641 agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat
     2701 gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     2761 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     2821 atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg
     2881 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2941 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     3001 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3061 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3121 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     3181 gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg aggatcgttt
     3241 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     3301 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     3361 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     3421 ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     3481 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     3541 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     3601 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     3661 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     3721 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc
     3781 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     3841 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     3901 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     3961 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     4021 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     4081 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     4141 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     4201 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
     4261 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
     4321 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
     4381 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
     4441 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
     4501 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     4561 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4621 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4681 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4741 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     4801 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4861 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4921 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4981 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     5041 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     5101 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     5161 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     5221 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     5281 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     5341 ccatgcat
//
