LOCUS       sequence-379348-        6214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of the genetically encoded calcium sensor 
            (GECI) G-Ca-FLITS to mScarlet-I fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6214)
  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 6214)
  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: pFR-mScarletI-antiFRET-P2A-G-Ca-FLITS
FEATURES             Location/Qualifiers
     source          1..6214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(212..231)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    256..303
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      632..1220
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1121..1140
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1368..1671
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1673..1875
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1826..1846
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2078..2100
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2162..2181
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2223..2341
                     /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             2416..2433
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2443..2460
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2479..2488
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2485..3180
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2618..2636)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2886..2905
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3301..3357
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3397..3453
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             3478..3726
                     /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(3790..3811)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4051..4070)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             4201..4644
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTFDFPEFLTMMARKMNDTDSEEEIREAFRVFDKDGNGYIGAAEL
                     RHVMTDLGEKLTDEEVDEMIRVADIDGDGQVNYEEFVQMMTAK"
     primer_bind     complement(4737..4756)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4802..4857
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4856..4875)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4945..4992
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        5018..5122
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5147..5167)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5191..5401
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        5213..5409
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5260..5395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(5444..6214,1..24)
                     /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(5444..6214,1..24)
                     /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(5498..5517)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6108..6127
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
       61 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
      121 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
      181 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
      241 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
      301 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
      361 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
      421 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
      481 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
      541 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
      601 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
      661 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
      721 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
      781 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
      841 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
      901 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
      961 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     1021 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     1081 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     1141 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     1201 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     1261 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgaggccggc ctattaatag
     1321 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     1381 acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg
     1441 acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat
     1501 ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct
     1561 attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg
     1621 gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat gctgatgcgg
     1681 ttttggcagt acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc
     1741 caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa
     1801 tgtcgtaata accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc
     1861 tatataagca gacgtcgttt agtgaaccgt cagatcacta gatgctttat tgcggtagtt
     1921 tatcacagtt aaattgctaa cgccagtctc gaacttaacg tgcagaagtt ggtcgtgagg
     1981 cactgggcag gtaagtatcg ggccctttgt gcggggggag cggctcgggg ctgtccgcgg
     2041 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     2101 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     2161 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattgga tcggaccgaa
     2221 atcaccacaa ttcagcaaat tgtgaacatc atcacgttca tctttccctg gttgccaaag
     2281 gcccattttc ctgtcagtaa cgagaaggtc gcgaattcag gcgcttttta gactggtcgt
     2341 agggagacca caacggtttc cctctagaaa taattttgtt taactataag aaggagatat
     2401 accatgcggg gttctcatca tcaccaccat cacagcagcg gcctggtgcc gcgcggcagc
     2461 tcgctagcgc tgccggtcgc caccatggtg agcaagggcg aggcagtgat caaggagttc
     2521 atgcggttca aggtgcacat ggagggctcc atgaacggcc acgagttcga gatcgagggc
     2581 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     2641 ggccccctgc ccttctcctg ggacatcctg tcccctcagt tcatgtacgg ctccagggcc
     2701 ttcatcaagc accccgccga catccccgac tactataagc agtccttccc cgagggcttc
     2761 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgccg tgaccgtgac ccaggacacc
     2821 tccctggagg acggcaccct gatctacaag gtgaagctcc gcggcaccaa cttccctcct
     2881 gacggccccg taatgcagaa gaagacaatg ggctgggaag cgtccaccga gcggttgtac
     2941 cccgaggacg gcgtgctgaa gggcgacatt aagatggccc tgcgcctgaa ggacggcggc
     3001 cgctacctgg cggacttcaa gaccacctac aaggccaaga agcccgtgca gatgcccggc
     3061 gcctacaacg tcgaccgcaa gttggacatc acctcccaca acgaggacta caccgtggtg
     3121 gaacagtacg aacgctccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     3181 tcgaccagat ctctggccga ggcggcagct aaggaggccg cggcgaagga ggcagccgcg
     3241 aaagaagcag ccgcaaagga agcggccgcc aaggctgctg caagatctct cggatcgggt
     3301 gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggaccttcg
     3361 ctagcgctgg atcccgccac aatggtcgac tcttcacgtc gtaagtggaa taaggcaggt
     3421 cacgcagtca gagctatagg tcggctgagc tcacccgtgg ctgtctatat caccgccgac
     3481 aagcagaaga acggcatcaa ggccaacttc aagatccgcc acaacatcga ggacggcggc
     3541 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     3601 cccgacaacc actacctgag ctaccagtcc aagctgagca aagaccccaa cgagaagcgc
     3661 gatcacatgg tcctactgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     3721 ctgtaccagg gtggcagcgg tggcatggtg agcaagggcg aggagctgtt caccggggtg
     3781 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     3841 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     3901 aagctgcccg tgccctggcc caccctcgtg accaccctgt cctggggcgt gcagtgcttc
     3961 gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     4021 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     4081 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     4141 gaggacggca acatcctggg gcacaagctg gagtacaact actatagcga cgatacgcgt
     4201 gaccaactga ctgaagagca gatcgcagaa tttaaagagg ctttctccct atttgacaag
     4261 gacggggatg ggacgataac aaccaaggag ctggggacgg tgatgcggtc tctggggcag
     4321 aaccccacag aagcagagct gcaggacatg atcaatgaag tagatgccga cggtgacggc
     4381 acattcgact tccctgagtt cctgacgatg atggcaagaa aaatgaatga cacagacagt
     4441 gaagaggaaa ttagagaagc gttccgcgtg tttgataagg acggcaatgg ctacatcggc
     4501 gcagcagagc ttcgccacgt gatgacagac cttggagaga agttaacaga tgaggaggtt
     4561 gatgaaatga tcagggtagc agacatcgat ggggatggtc aggtaaacta cgaagagttt
     4621 gtacaaatga tgacagcgaa gtagaagctt gtgattaacc tcaggtgcag gctgcctatc
     4681 agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga gatcgatctt
     4741 tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc
     4801 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     4861 ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt
     4921 ttggcaacat atgcccatat gtaactagca taaccccttg gggcctctaa acgggtcttg
     4981 aggggttttt tgctgaaagc atgctcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5041 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5101 ataatattga aaaaggaaga gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     5161 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     5221 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     5281 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     5341 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     5401 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     5461 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     5521 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     5581 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     5641 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     5701 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     5761 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     5821 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     5881 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     5941 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     6001 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     6061 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     6121 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     6181 cgccgctccc gattcgcagc gcatcgcctt ctat
//
