LOCUS       sequence-260214-        5319 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 5319)
  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 5319)
  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: Lck-cpmTq2-Calcium-lifetime-sensor
FEATURES             Location/Qualifiers
     source          1..5319
                     /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"
     regulatory      595..604
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      640..649
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             661..717
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             742..990
                     /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(1054..1075)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1315..1334)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    1918..1983
                     /label=MCS
                     /note="multiple cloning site"
     polyA_signal    2107..2228
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2144..2163)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2198..2217
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2235..2690)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2372..2393)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2584..2603
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2717..2821
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2823..3180
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2823..3014
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2846..2866)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3031..3166
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3215..4009
                     /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             3215..4009
                     /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(3269..3288)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3879..3898
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4197..4216)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4241..4288
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4617..5205
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5106..5125
                     /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 gctagccacc
      601 atgggctgcg tgtgcagcag caaccccgag ctaccggtcg ccaccatggt cgactcttca
      661 cgtcgtaagt ggaataaggc aggtcacgca gtcagagcta taggtcggct gagctcaccc
      721 gtggttgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
      781 cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc
      841 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg
      901 agcaaagacc ccaacgagaa gcgcgatcac atggtcctac tggagttcgt gaccgccgcc
      961 gggatcactc tcggcatgga cgagctgtac cagggtggca gcggtggcat ggtgagcaag
     1021 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1081 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     1141 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1201 ctgtcctggg gcgtgcagtg cttcgcccgc taccccgacc acatgaagca gcacgacttc
     1261 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     1321 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     1381 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     1441 aactactata gcgacaacac gcgtgaccaa ctgactgaag agcagatcgc agaatttaaa
     1501 gaggctttct ccctatttga caaggacggg gatgggacga taacaaccaa ggagctgggg
     1561 acggtgatgc ggtctctggg gcagaacccc acagaagcag agctgcagga catgatcaat
     1621 gaagtagatg ccgacggtga cggcacattc gacttccctg agttcctgac gatgatggca
     1681 agaaaaatga atgacacaga cagtgaagag gaaattagag aagcgttccg cgtgtttgat
     1741 aaggacggca atggctacat cggcgcagca gagcttcgcc acgtgatgac agaccttgga
     1801 gagaagttaa cagatgagga ggttgatgaa atgatcaggg tagcagacat cgatggggat
     1861 ggtcaggtaa actacgaaga gtttgtccaa atgatgacag cgaagtaggt gtacaagtcc
     1921 ggactcagat ctcgagctca agcttcgaat tctgcagtcg acggtaccgc gggcccggga
     1981 tccaccggat ctagataact gatcataatc agccatacca catttgtaga ggttttactt
     2041 gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt
     2101 gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2161 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2221 gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     2281 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     2341 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     2401 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     2461 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     2521 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     2581 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     2641 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac
     2701 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     2761 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     2821 tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     2881 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     2941 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3001 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3061 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3121 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3181 gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc
     3241 aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat
     3301 cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt
     3361 caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg
     3421 gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag
     3481 ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc
     3541 tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc
     3601 tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga
     3661 agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga
     3721 actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg
     3781 cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg
     3841 tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc
     3901 tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc
     3961 cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg
     4021 gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc
     4081 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc
     4141 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa
     4201 acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat
     4261 aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac
     4321 tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc
     4381 ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca
     4441 ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt
     4501 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4561 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4621 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4681 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4741 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4801 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4861 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4921 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4981 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     5041 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     5101 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     5161 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     5221 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     5281 tcccctgatt ctgtggataa ccgtattacc gccatgcat
//
