LOCUS       sequence-379341-        5317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial isolation of 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 5317)
  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 5317)
  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: pFPO-His-G-Ca-FLITS
FEATURES             Location/Qualifiers
     source          1..5317
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(215..234)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    259..306
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      635..1223
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1124..1143
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1371..1674
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1676..1878
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1829..1849
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2081..2103
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2165..2184
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2226..2344
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             2419..2436
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2446..2463
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2503..2559
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             2584..2832
                     /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(2896..2917)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3157..3176)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3307..3750
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTFDFPEFLTMMARKMNDTDSEEEIREAFRVFDKDGNGYIGAAEL
                     RHVMTDLGEKLTDEEVDEMIRVADIDGDGQVNYEEFVQMMTAK"
     primer_bind     complement(3843..3862)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3908..3963
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3962..3981)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4051..4098
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4124..4228
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4253..4273)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4297..4507
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        4319..4515
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4366..4501
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(4550..5317,1..27)
                     /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             join(4550..5317,1..27)
                     /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(4604..4623)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5214..5233
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag
       61 cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg
      121 gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc
      181 tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg
      241 aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt
      301 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
      361 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
      421 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcctcaggtt
      481 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
      541 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
      601 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
      661 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
      721 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
      781 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
      841 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
      901 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
      961 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     1021 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     1081 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     1141 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     1201 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     1261 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgaggcc ggcctattaa
     1321 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     1381 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     1441 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     1501 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtccgccc
     1561 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     1621 cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catgctgatg
     1681 cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg atttccaagt
     1741 ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca
     1801 aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt acggtgggag
     1861 gtctatataa gcagacgtcg tttagtgaac cgtcagatca ctagatgctt tattgcggta
     1921 gtttatcaca gttaaattgc taacgccagt ctcgaactta acgtgcagaa gttggtcgtg
     1981 aggcactggg caggtaagta tcgggccctt tgtgcggggg gagcggctcg gggctgtccg
     2041 cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg
     2101 accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt cctacagctc
     2161 ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt ggatcggacc
     2221 gaaatcacca caattcagca aattgtgaac atcatcacgt tcatctttcc ctggttgcca
     2281 aaggcccatt ttcctgtcag taacgagaag gtcgcgaatt caggcgcttt ttagactggt
     2341 cgtagggaga ccacaacggt ttccctctag aaataatttt gtttaactat aagaaggaga
     2401 tataccatgc ggggttctca tcatcaccac catcacagca gcggcctggt gccgcgcggc
     2461 agctcgctag cgctggatcc cgccacaatg gtcgactctt cacgtcgtaa gtggaataag
     2521 gcaggtcacg cagtcagagc tataggtcgg ctgagctcac ccgtggctgt ctatatcacc
     2581 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
     2641 ggcggcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2701 ctgctgcccg acaaccacta cctgagctac cagtccaagc tgagcaaaga ccccaacgag
     2761 aagcgcgatc acatggtcct actggagttc gtgaccgccg ccgggatcac tctcggcatg
     2821 gacgagctgt accagggtgg cagcggtggc atggtgagca agggcgagga gctgttcacc
     2881 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2941 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     3001 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgtcctg gggcgtgcag
     3061 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     3121 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     3181 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     3241 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacta tagcgacgat
     3301 acgcgtgacc aactgactga agagcagatc gcagaattta aagaggcttt ctccctattt
     3361 gacaaggacg gggatgggac gataacaacc aaggagctgg ggacggtgat gcggtctctg
     3421 gggcagaacc ccacagaagc agagctgcag gacatgatca atgaagtaga tgccgacggt
     3481 gacggcacat tcgacttccc tgagttcctg acgatgatgg caagaaaaat gaatgacaca
     3541 gacagtgaag aggaaattag agaagcgttc cgcgtgtttg ataaggacgg caatggctac
     3601 atcggcgcag cagagcttcg ccacgtgatg acagaccttg gagagaagtt aacagatgag
     3661 gaggttgatg aaatgatcag ggtagcagac atcgatgggg atggtcaggt aaactacgaa
     3721 gagtttgtac aaatgatgac agcgaagtag aagcttgtga ttaacctcag gtgcaggctg
     3781 cctatcagaa ggtggtggct ggtgtggcca atgccctggc tcacaaatac cactgagatc
     3841 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     3901 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     3961 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     4021 tagagtttgg caacatatgc ccatatgtaa ctagcataac cccttggggc ctctaaacgg
     4081 gtcttgaggg gttttttgct gaaagcatgc tcggggaaat gtgcgcggaa cccctatttg
     4141 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4201 gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg
     4261 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4321 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4381 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4441 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4501 ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     4561 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     4621 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     4681 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aagacgaggc
     4741 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     4801 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     4861 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     4921 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     4981 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     5041 gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg gcgaggatct
     5101 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     5161 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     5221 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     5281 cggtatcgcc gctcccgatt cgcagcgcat cgccttc
//
