LOCUS       sequence-379340-        6202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of the genetically encoded calcium sensor 
            (GECI) GCaMP3 to mScarlet-I fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6202)
  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 6202)
  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-GCaMP3
FEATURES             Location/Qualifiers
     source          1..6202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(210..229)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    254..301
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      630..1218
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1119..1138
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1366..1669
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1671..1873
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1824..1844
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2076..2098
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2160..2179
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2221..2339
                     /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             2414..2431
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2441..2458
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2477..2486
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2483..3178
                     /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(2616..2634)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2884..2903
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3299..3355
                     /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             3395..3451
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     3664..3685
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3791..3812)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4052..4071)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             4187..4630
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAEL
                     RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     complement(4723..4742)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4788..4843
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4842..4861)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4931..4978
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        5004..5108
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5133..5153)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5177..5387
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        5199..5395
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5246..5381
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(5430..6202,1..22)
                     /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(5430..6202,1..22)
                     /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(5484..5503)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6094..6113
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg
       61 cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
      121 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
      181 ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat accggaagga
      241 acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt
      301 cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat
      361 tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa
      421 ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca
      481 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
      541 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
      601 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
      661 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
      721 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
      781 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
      841 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
      901 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
      961 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     1021 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     1081 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     1141 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     1201 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     1261 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg aggccggcct attaatagta
     1321 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     1381 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     1441 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     1501 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc cgccccctat
     1561 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttacggga
     1621 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc tgatgcggtt
     1681 ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     1741 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     1801 tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     1861 tataagcaga cgtcgtttag tgaaccgtca gatcactaga tgctttattg cggtagttta
     1921 tcacagttaa attgctaacg ccagtctcga acttaacgtg cagaagttgg tcgtgaggca
     1981 ctgggcaggt aagtatcggg ccctttgtgc ggggggagcg gctcggggct gtccgcgggg
     2041 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     2101 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg
     2161 caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc ggaccgaaat
     2221 caccacaatt cagcaaattg tgaacatcat cacgttcatc tttccctggt tgccaaaggc
     2281 ccattttcct gtcagtaacg agaaggtcgc gaattcaggc gctttttaga ctggtcgtag
     2341 ggagaccaca acggtttccc tctagaaata attttgttta actataagaa ggagatatac
     2401 catgcggggt tctcatcatc accaccatca cagcagcggc ctggtgccgc gcggcagctc
     2461 gctagcgctg ccggtcgcca ccatggtgag caagggcgag gcagtgatca aggagttcat
     2521 gcggttcaag gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga
     2581 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     2641 ccccctgccc ttctcctggg acatcctgtc ccctcagttc atgtacggct ccagggcctt
     2701 catcaagcac cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa
     2761 gtgggagcgc gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc
     2821 cctggaggac ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga
     2881 cggccccgta atgcagaaga agacaatggg ctgggaagcg tccaccgagc ggttgtaccc
     2941 cgaggacggc gtgctgaagg gcgacattaa gatggccctg cgcctgaagg acggcggccg
     3001 ctacctggcg gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc
     3061 ctacaacgtc gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga
     3121 acagtacgaa cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagtc
     3181 gaccagatct ctggccgagg cggcagctaa ggaggccgcg gcgaaggagg cagccgcgaa
     3241 agaagcagcc gcaaaggaag cggccgccaa ggctgctgca agatctctcg gatcgggtgc
     3301 tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg gaccttcgct
     3361 agcgctggat cccgccacaa tggtcgactc atcacgtcgt aagtggaata agacaggtca
     3421 cgcagtcaga gctataggtc ggctgagctc actcgagaac gtctatatca aggccgacaa
     3481 gcagaagaac ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcggcgt
     3541 gcagctcgcc taccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     3601 cgacaaccac tacctgagcg tgcagtccaa actttcgaaa gaccccaacg agaagcgcga
     3661 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     3721 gtacaagggc ggtaccggag ggagcatggt gagcaagggc gaggagctgt tcaccggggt
     3781 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     3841 cgagggtgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     3901 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     3961 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     4021 ctacatccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     4081 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     4141 ggaggacggc aacatcctgg ggcacaagct ggagtacaac acgcgtgacc aactgactga
     4201 agagcagatc gcagaattta aagaggcttt ctccctattt gacaaggacg gggatgggac
     4261 aataacaacc aaggagctgg ggacggtgat gcggtctctg gggcagaacc ccacagaagc
     4321 agagctgcag gacatgatca atgaagtaga tgccgacggt gacggcacaa tcgacttccc
     4381 tgagttcctg acaatgatgg caagaaaaat gaaagacaca gacagtgaag aagaaattag
     4441 agaagcgttc cgtgtgtttg ataaggatgg caatggctac atcagtgcag cagagcttcg
     4501 ccacgtgatg acaaaccttg gagagaagtt aacagatgaa gaggttgatg aaatgatcag
     4561 ggaagcagac atcgatgggg atggtcaggt aaactacgaa gagtttgtac aaatgatgac
     4621 agcgaagtag aagcttgtga ttaacctcag gtgcaggctg cctatcagaa ggtggtggct
     4681 ggtgtggcca atgccctggc tcacaaatac cactgagatc gatctttttc cctctgccaa
     4741 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     4801 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     4861 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     4921 ccatatgtaa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     4981 gaaagcatgc tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5041 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5101 ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     5161 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     5221 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     5281 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     5341 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg
     5401 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     5461 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     5521 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     5581 ccgacctgtc cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg
     5641 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     5701 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     5761 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     5821 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     5881 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     5941 tcgccaggct caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     6001 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     6061 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     6121 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     6181 cgcagcgcat cgccttctat cg
//
