LOCUS       sequence-379335-        6298 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of the genetically encoded calcium sensor 
            (GECI) R-GECO1 to mTurquoise2 fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6298)
  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 6298)
  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-mTq2-antiFRET-P2A-R-GECO1
FEATURES             Location/Qualifiers
     source          1..6298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(188..207)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    232..279
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      608..1196
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1097..1116
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1344..1647
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1649..1851
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1802..1822
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2054..2076
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2138..2157
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2199..2317
                     /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             2392..2409
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2419..2436
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2455..2464
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2461..2463,2464..2466,2467..3177)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2506..2527)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2767..2786)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3114..3135
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3298..3354
                     /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             3394..3450
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     primer_bind     4146..4165
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4189..4632
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTFDFPEFLTMMARKMNDTDSEEEIREAFRVFDKDGNGYIGAAEL
                     RHVMTDLGEKLTDEEVDEMIRVADIDGDGQVNYEEFVQMMTAK"
     primer_bind     complement(4725..4744)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4790..4845
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4844..4863)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4933..4980
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        5006..5110
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5112..5469
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5112..5303
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5135..5155)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5320..5455
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5504..6298
                     /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             5504..6298
                     /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(5558..5577)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6168..6187
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
       61 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
      121 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga
      181 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
      241 aaagacagaa taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc
      301 agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt
      361 cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg
      421 tcggggcggc aggccctgcc atagcctcag gttactcata tatactttag attgatttaa
      481 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
      541 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
      601 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
      661 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
      721 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
      781 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
      841 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
      901 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
      961 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     1021 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     1081 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     1141 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     1201 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     1261 ttcctgcgtt atcccctgag gccggcctat taatagtaat caattacggg gtcattagtt
     1321 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     1381 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     1441 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     1501 gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg
     1561 cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc
     1621 tacgtattag tcatcgctat taccatgctg atgcggtttt ggcagtacac caatgggcgt
     1681 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     1741 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg
     1801 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagacg tcgtttagtg
     1861 aaccgtcaga tcactagatg ctttattgcg gtagtttatc acagttaaat tgctaacgcc
     1921 agtctcgaac ttaacgtgca gaagttggtc gtgaggcact gggcaggtaa gtatcgggcc
     1981 ctttgtgcgg ggggagcggc tcggggctgt ccgcgggggg acggctgcct tcggggggga
     2041 cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc ctctgctaac
     2101 catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt tattgtgctg
     2161 tctcatcatt ttggcaaaga attggatcgg accgaaatca ccacaattca gcaaattgtg
     2221 aacatcatca cgttcatctt tccctggttg ccaaaggccc attttcctgt cagtaacgag
     2281 aaggtcgcga attcaggcgc tttttagact ggtcgtaggg agaccacaac ggtttccctc
     2341 tagaaataat tttgtttaac tataagaagg agatatacca tgcggggttc tcatcatcac
     2401 caccatcaca gcagcggcct ggtgccgcgc ggcagctcgc tagcgctgcc ggtcgccacc
     2461 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     2521 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     2581 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
     2641 ctcgtgacca ccctgtcctg gggcgtgcag tgcttcgccc gctaccccga ccacatgaag
     2701 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     2761 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     2821 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     2881 aagctggagt acaactactt tagcgacaac gtctatatca ccgccgacaa gcagaagaac
     2941 ggcatcaagg ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc
     3001 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     3061 tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     3121 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcg
     3181 accagatctc tggccgaggc ggcagctaag gaggccgcgg cgaaggaggc agccgcgaaa
     3241 gaagcagccg caaaggaagc ggccgccaag gctgctgcaa gatctctcgg atcgggtgct
     3301 actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg accttcgcta
     3361 gcgctggatc ccgccacaat ggtcgactca tcacgtcgta agtggaataa ggcaggtcac
     3421 gcagtcagag ctataggtcg gctgagctca cccgtggttt ccgagcggat gtaccccgag
     3481 gacggcgccc tgaagagcga gatcaagaag gggctgaggc tgaaggacgg cggccactac
     3541 gccgccgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc cggcgcctac
     3601 atcgtcgaca tcaagttgga catcgtgtcc cacaacgagg actacaccat cgtggaacag
     3661 tgcgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta caagggaggt
     3721 acaggcggga gtctggtgag caagggcgag gaggataaca tggccatcat caaggagttc
     3781 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     3841 gagggcgagg gccgccccta cgaggccttt cagaccgcta agctgaaggt gaccaagggt
     3901 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
     3961 tacattaagc acccagccga catccccgac tacttcaagc tgtccttccc cgagggcttc
     4021 aggtgggagc gcgtgatgaa cttcgaggac ggcggcatta ttcacgttaa ccaggactcc
     4081 tccctgcagg acggcgtatt catctacaag gtgaagctgc gcggcaccaa cttccccccc
     4141 gacggccccg taatgcagaa gaagaccatg ggctgggagg ctacgcgtga ccaactgact
     4201 gaagagcaga tcgcagaatt taaagaggct ttctccctat ttgacaagga cggggatggg
     4261 acgataacaa ccaaggagct ggggacggtg atgcggtctc tggggcagaa ccccacagaa
     4321 gcagagctgc aggacatgat caatgaagta gatgccgacg gtgacggcac attcgacttc
     4381 cctgagttcc tgacgatgat ggcaagaaaa atgaatgaca cagacagtga agaggaaatt
     4441 agagaagcgt tccgcgtgtt tgataaggac ggcaatggct acatcggcgc agcagagctt
     4501 cgccacgtga tgacagacct tggagagaag ttaacagatg aggaggttga tgaaatgatc
     4561 agggtagcag acatcgatgg ggatggtcag gtaaactacg aagagtttgt acaaatgatg
     4621 acagcgaagt agaagcttgt gattaacctc aggtgcaggc tgcctatcag aaggtggtgg
     4681 ctggtgtggc caatgccctg gctcacaaat accactgaga tcgatctttt tccctctgcc
     4741 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     4801 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     4861 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     4921 gcccatatgt aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     4981 ctgaaagcat gctcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     5041 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     5101 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     5161 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     5221 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     5281 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     5341 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     5401 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     5461 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     5521 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     5581 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     5641 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     5701 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     5761 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     5821 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     5881 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     5941 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     6001 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     6061 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     6121 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     6181 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     6241 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctga
//
