LOCUS       sequence-383617-        6271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of the genetically encoded calcium sensor 
            (GECI) RCaMP1h to mTurquoise2 fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6271)
  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 6271)
  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-RCaMP1h
FEATURES             Location/Qualifiers
     source          1..6271
                     /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     1816..1836
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     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="RRKWNKTGHAVRAIGRLSS"
     primer_bind     complement(4712..4731)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4777..4832
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4831..4850)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4920..4967
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        4993..5097
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5099..5456
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5099..5290
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5122..5142)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5307..5442
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(5491..6271,1..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(5491..6271,1..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(5545..5564)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6155..6174
                     /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 ataagacagg tcacgcagtc agagctatag gtcggctgag ctcagcgatc aacaccgaaa
     3481 tgatgtaccc agcggatggt ggtctgcgtg gttacactca catggcgctg aaagttgatg
     3541 gcggcggtca cctgtcctgt tctttcgtga ccacctaccg ctccaaaaag actgtcggca
     3601 acattaagat gcctgccatt cattacgtca gccaccgtct ggagcgcctg gaggagagcg
     3661 ataacgaaat gtttgtcgta cagcgtgaac acgcagttgc caagtttgtg ggcctgggtg
     3721 gtggcggcgg taccggaggg agcatgaact ccctgatcaa ggagaacatg cgtatgaaag
     3781 tggttctgga aggctccgta aacggccacc agttcaaatg cactggtgaa ggcgaaggca
     3841 acccgtatat gggcacccag actatgcgta tcaaagtgat cgagggtggt ccgctgccgt
     3901 ttgcgttcga catcctggcg acgtccttta tgtatggctc ccgtaccttc atcaaatatc
     3961 cgaaaggcat cccggatttc tttaagcagt ccttcccgga aggttttacc tgggaacgtg
     4021 tgacccgtta cgaagacggc ggcgtaatta ccgttatgca agacacgtct ctggaggatg
     4081 gctgcctggt gtatcacgtg caggttcgcg gtgttaactt cccgagcaat ggtgctgtaa
     4141 tgcaaaagaa aaccaaaggt tgggagccta cggactccca actgactgaa gagcagatcg
     4201 cagaatttaa agaggctttc tccctatttg acaaggacgg ggatgggaca ataacaacca
     4261 aggagctggg gacggtgatg cggtctctgg ggcagaaccc cacagaagca gagctgcagg
     4321 acatgatcaa tgaagtagat gccgacggtg acggcacaat cgacttccct gagttcctga
     4381 ttatgatggc aagaaaaatg aaatacacag acagtgaaga agaaattaga gaagcgttcg
     4441 gcgtgtttga taaggatggc aatggctaca tcagtgcagc agagcttcgc cacgtgatga
     4501 caaaccttgg agagaagtta acagatgaag aggttgatga aatgatcagg gaagcagaca
     4561 gcgatgggga tggtcaggta aactacgaag agtttgtaca aatgatgaca gcgaagtaga
     4621 agcttgtgat taacctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa
     4681 tgccctggct cacaaatacc actgagatcg atctttttcc ctctgccaaa aattatgggg
     4741 acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt attttcattg
     4801 caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg agggcaaatc
     4861 atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc catatgtaac
     4921 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaagcatgct
     4981 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5041 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct
     5101 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     5161 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     5221 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     5281 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     5341 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     5401 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc
     5461 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     5521 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     5581 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     5641 accgacctgt ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg
     5701 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     5761 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     5821 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     5881 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     5941 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     6001 ttcgccaggc tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     6061 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     6121 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     6181 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     6241 tcgcagcgca tcgccttcta tcgccttctt g
//
