LOCUS       sequence-377501-        6202 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of the genetically encoded calcium sensor 
            (GECI) jGCaMP7c 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-jGCaMP7c
FEATURES             Location/Qualifiers
     source          1..6202
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(206..225)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    250..297
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      626..1214
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1115..1134
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1362..1665
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1667..1869
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1820..1840
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2072..2094
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2156..2175
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2217..2335
                     /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             2410..2427
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2437..2454
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2473..2482
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2479..3174
                     /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(2612..2630)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2880..2899
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3295..3351
                     /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             3391..3447
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     3660..3681
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3787..3808)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4048..4067)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(4719..4738)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4784..4839
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4838..4857)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4927..4974
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        5000..5104
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5129..5149)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5173..5383
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        5195..5391
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5242..5377
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(5426..6202,1..18)
                     /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(5426..6202,1..18)
                     /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(5480..5499)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6090..6109
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
       61 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
      121 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
      181 tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc
      241 gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg tttgttcata
      301 aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg
      361 gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc
      421 cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt tactcatata
      481 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
      541 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
      601 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
      661 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
      721 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
      781 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
      841 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
      901 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
      961 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     1021 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     1081 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     1141 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     1201 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     1261 cttttgctca catgttcttt cctgcgttat cccctgaggc cggcctatta atagtaatca
     1321 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
     1381 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
     1441 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
     1501 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtccgcc ccctattgac
     1561 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt acgggacttt
     1621 cctacttggc agtacatcta cgtattagtc atcgctatta ccatgctgat gcggttttgg
     1681 cagtacacca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
     1741 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
     1801 aataaccccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
     1861 agcagacgtc gtttagtgaa ccgtcagatc actagatgct ttattgcggt agtttatcac
     1921 agttaaattg ctaacgccag tctcgaactt aacgtgcaga agttggtcgt gaggcactgg
     1981 gcaggtaagt atcgggccct ttgtgcgggg ggagcggctc ggggctgtcc gcggggggac
     2041 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc
     2101 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac
     2161 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tggatcggac cgaaatcacc
     2221 acaattcagc aaattgtgaa catcatcacg ttcatctttc cctggttgcc aaaggcccat
     2281 tttcctgtca gtaacgagaa ggtcgcgaat tcaggcgctt tttagactgg tcgtagggag
     2341 accacaacgg tttccctcta gaaataattt tgtttaacta taagaaggag atataccatg
     2401 cggggttctc atcatcacca ccatcacagc agcggcctgg tgccgcgcgg cagctcgcta
     2461 gcgctgccgg tcgccaccat ggtgagcaag ggcgaggcag tgatcaagga gttcatgcgg
     2521 ttcaaggtgc acatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc
     2581 gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa gggtggcccc
     2641 ctgcccttct cctgggacat cctgtcccct cagttcatgt acggctccag ggccttcatc
     2701 aagcaccccg ccgacatccc cgactactat aagcagtcct tccccgaggg cttcaagtgg
     2761 gagcgcgtga tgaacttcga ggacggcggc gccgtgaccg tgacccagga cacctccctg
     2821 gaggacggca ccctgatcta caaggtgaag ctccgcggca ccaacttccc tcctgacggc
     2881 cccgtaatgc agaagaagac aatgggctgg gaagcgtcca ccgagcggtt gtaccccgag
     2941 gacggcgtgc tgaagggcga cattaagatg gccctgcgcc tgaaggacgg cggccgctac
     3001 ctggcggact tcaagaccac ctacaaggcc aagaagcccg tgcagatgcc cggcgcctac
     3061 aacgtcgacc gcaagttgga catcacctcc cacaacgagg actacaccgt ggtggaacag
     3121 tacgaacgct ccgagggccg ccactccacc ggcggcatgg acgagctgta caagtcgacc
     3181 agatctctgg ccgaggcggc agctaaggag gccgcggcga aggaggcagc cgcgaaagaa
     3241 gcagccgcaa aggaagcggc cgccaaggct gctgcaagat ctctcggatc gggtgctact
     3301 aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc ttcgctagcg
     3361 ctggatcccg ccacaatggt cgactcatca cgtcgtaagt ggaataagac aggtcacgca
     3421 gtcagagcta taggtcggct gagctcacag cctaacgtct atatcaaggc cgacaagcag
     3481 aagaacggca tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag
     3541 ctcgcctacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     3601 aaccactacc tgagcgtgca gtccaaactt tcgaaagacc ccaacgagaa gcgcgatcac
     3661 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     3721 aagggcggta ccggagggag catggtgagc aagggcgagg agctgttcac cggggtggtg
     3781 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     3841 ggtgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     3901 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
     3961 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     4021 atccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     4081 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     4141 gacggcaaca tcctggggca caagctggag tacaaccttc ctgaccaact gactgaagag
     4201 cagatcgcag aatttaaaga ggctttctcc ctatttgaca aggacgggga tgggacaata
     4261 acaaccaagg agctggggac ggtgatgcgt tctctggggc agaaccccac agaagcagag
     4321 ctgcaggaca tgatcaatga agtagatgcc gacggtgacg gcacaatcga cttccctgag
     4381 ttcctgacaa tgatggcaag aaaagggagc tacagggaca gtgaagaaga aattagagaa
     4441 gcgttcggtg tgtttgataa ggatggcaat ggctacatca gtgcagcaga gcttcgccac
     4501 gtgatgaaca accttggaga gaagttaaca gatgaagagg ttgatgaaat gatcagggaa
     4561 gcagacatcg atggggatgg tcaggtaaac tacgaagagt ttgtacaaat gatgacagcg
     4621 aagtagaagc ttgtgattaa cctcaggtgc aggctgccta tcagaaggtg gtggctggtg
     4681 tggccaatgc cctggctcac aaataccact gagatcgatc tttttccctc tgccaaaaat
     4741 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     4801 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg
     4861 gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat
     4921 atgtaactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     4981 gcatgctcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5041 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5101 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     5161 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     5221 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5281 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     5341 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     5401 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     5461 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     5521 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     5581 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     5641 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     5701 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     5761 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     5821 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     5881 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     5941 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     6001 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     6061 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     6121 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     6181 gcgcatcgcc ttctatcgcc tt
//
