LOCUS       sequence-377511-        6238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of the genetically encoded calcium sensor 
            (GECI) CH-GECO2.1 to mTurquoise2 fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6238)
  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 6238)
  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-CH-GECO2.1
FEATURES             Location/Qualifiers
     source          1..6238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(202..221)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    246..293
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      622..1210
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1111..1130
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1358..1661
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1663..1865
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2068..2090
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2152..2171
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2213..2331
                     /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             2406..2423
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2433..2450
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2469..2478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2475..2477,2478..2480,2481..3191)
                     /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(2520..2541)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2781..2800)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3128..3149
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3312..3368
                     /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             3408..3464
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     primer_bind     complement(3901..3919)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4169..4188
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4751..4770)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4816..4871
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4870..4889)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4959..5006
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        5032..5136
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5161..5181)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5205..5415
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        5227..5423
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5274..5409
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(5458..6238,1..14)
                     /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(5458..6238,1..14)
                     /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(5512..5531)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6122..6141
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
       61 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
      121 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
      181 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
      241 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
      301 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
      361 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
      421 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
      481 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
      541 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
      601 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
      661 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
      721 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
      781 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
      841 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
      901 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
      961 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     1021 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     1081 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     1141 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     1201 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1261 tgctcacatg ttctttcctg cgttatcccc tgaggccggc ctattaatag taatcaatta
     1321 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1381 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1441 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1501 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
     1561 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
     1621 cttggcagta catctacgta ttagtcatcg ctattaccat gctgatgcgg ttttggcagt
     1681 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1741 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
     1801 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1861 gacgtcgttt agtgaaccgt cagatcacta gatgctttat tgcggtagtt tatcacagtt
     1921 aaattgctaa cgccagtctc gaacttaacg tgcagaagtt ggtcgtgagg cactgggcag
     1981 gtaagtatcg ggccctttgt gcggggggag cggctcgggg ctgtccgcgg ggggacggct
     2041 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     2101 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     2161 tggttattgt gctgtctcat cattttggca aagaattgga tcggaccgaa atcaccacaa
     2221 ttcagcaaat tgtgaacatc atcacgttca tctttccctg gttgccaaag gcccattttc
     2281 ctgtcagtaa cgagaaggtc gcgaattcag gcgcttttta gactggtcgt agggagacca
     2341 caacggtttc cctctagaaa taattttgtt taactataag aaggagatat accatgcggg
     2401 gttctcatca tcaccaccat cacagcagcg gcctggtgcc gcgcggcagc tcgctagcgc
     2461 tgccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     2521 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     2581 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     2641 tgccctggcc caccctcgtg accaccctgt cctggggcgt gcagtgcttc gcccgctacc
     2701 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     2761 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2821 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2881 acatcctggg gcacaagctg gagtacaact actttagcga caacgtctat atcaccgccg
     2941 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggcg
     3001 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     3061 tgcccgacaa ccactacctg agcacccagt ccaagctgag caaagacccc aacgagaagc
     3121 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     3181 agctgtacaa gtcgaccaga tctctggccg aggcggcagc taaggaggcc gcggcgaagg
     3241 aggcagccgc gaaagaagca gccgcaaagg aagcggccgc caaggctgct gcaagatctc
     3301 tcggatcggg tgctactaac ttcagcctgc tgaagcaggc tggagacgtg gaggagaacc
     3361 ctggaccttc gctagcgctg gatcccgcca caatggtcga ctcatcacgt cgtaagtgga
     3421 ataaggcagg tcatgcagtc agagctatag gtcggctgag ctcacttgag tcgctgctgt
     3481 ccaccgagcg gctttacccc gaggacggcg ctctgaaggg cgagatcaag cagaggctga
     3541 agctgaagga cggcggccac tacgccgccg aggtcaagac cacctacaag gccaagaagc
     3601 ccgtgcagct gcccgacgcc tacatcgtcg acatcaagtt ggacatcgtg tcccacaacg
     3661 aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc accggcggca
     3721 tggacgagct gtacaagggt ggcactggcg gttccatggt gagcaagggc gtggaggata
     3781 acatggcctt catcaaggag ttcatgcgct tcaaggtgca catggagggc tccgtcaacg
     3841 gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg
     3901 ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc
     3961 agttcatgta cggctccaag gcctacatta agcacccagc cgacatcccc gactactgga
     4021 agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggaggtg
     4081 tggtgaccgt gacccaggac tcctccctgc aggacggcga gttcatctac aaggtgaagc
     4141 tgcgcggcac caacttcccc tccaacggcc ccgtaatgca gaagaagacc atgggctggc
     4201 tgggcggtac gcgtgaccaa ctgactgaag agcagatcgc agaatttaaa gaggctttct
     4261 ccctatttga caagggcggg gctgggacga taacaaccaa ggagctgggg acggtgatgc
     4321 ggtctctggg gcagaacccc acagaagcag agctgcagga catgatcaat gaagtagatg
     4381 ccgacggtga cggcacatta gacttccctg agttcctgac gatgatggca agaaaaatga
     4441 atgactcaga cagtgaagag gaaattagag aagcgttccg tgtgtttgat aaggacggca
     4501 atggctacat cggcgcagca gagcttcgcc acgtgatgac aaaccttgga gagaagttaa
     4561 cagatgaaga ggttgatgaa atgatcaggg tagcagacat cgatggggat ggtcaggtaa
     4621 actacgaaga gtttgtacaa atgatgacag cgaagtagaa gcttgtgatt aacctcaggt
     4681 gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca
     4741 ctgagatcga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc
     4801 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt
     4861 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt
     4921 atttggttta gagtttggca acatatgccc atatgtaact agcataaccc cttggggcct
     4981 ctaaacgggt cttgaggggt tttttgctga aagcatgctc ggggaaatgt gcgcggaacc
     5041 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     5101 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     5161 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     5221 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     5281 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     5341 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     5401 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     5461 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     5521 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     5581 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     5641 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     5701 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     5761 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     5821 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     5881 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     5941 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     6001 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     6061 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     6121 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     6181 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttg
//
