LOCUS       sequence-379338-        6286 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of the genetically encoded calcium sensor 
            (GECI) K-GECO1 to mTurquoise2 fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6286)
  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 6286)
  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-K-GECO1
FEATURES             Location/Qualifiers
     source          1..6286
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(194..213)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    238..285
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      614..1202
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1103..1122
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1350..1653
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1655..1857
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1808..1828
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2060..2082
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2144..2163
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2205..2323
                     /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             2398..2415
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2425..2442
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2461..2470
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2467..2469,2470..2472,2473..3183)
                     /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(2512..2533)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2773..2792)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3120..3141
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3304..3360
                     /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"
     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
     promoter        5106..5463
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5106..5297
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5129..5149)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5314..5449
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(5498..6286,1..6)
                     /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(5498..6286,1..6)
                     /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(5552..5571)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6162..6181
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
       61 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
      121 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
      181 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
      241 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
      301 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
      361 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
      421 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
      481 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
      541 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
      601 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
      661 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
      721 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
      781 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
      841 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
      901 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
      961 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     1021 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     1081 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     1141 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     1201 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     1261 tgttctttcc tgcgttatcc cctgaggccg gcctattaat agtaatcaat tacggggtca
     1321 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     1381 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     1441 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     1501 ttggcagtac atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt
     1561 aaatggcccg cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag
     1621 tacatctacg tattagtcat cgctattacc atgctgatgc ggttttggca gtacaccaat
     1681 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     1741 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa taaccccgcc
     1801 ccgttgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagacgtcgt
     1861 ttagtgaacc gtcagatcac tagatgcttt attgcggtag tttatcacag ttaaattgct
     1921 aacgccagtc tcgaacttaa cgtgcagaag ttggtcgtga ggcactgggc aggtaagtat
     1981 cgggcccttt gtgcgggggg agcggctcgg ggctgtccgc ggggggacgg ctgccttcgg
     2041 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct
     2101 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt
     2161 gtgctgtctc atcattttgg caaagaattg gatcggaccg aaatcaccac aattcagcaa
     2221 attgtgaaca tcatcacgtt catctttccc tggttgccaa aggcccattt tcctgtcagt
     2281 aacgagaagg tcgcgaattc aggcgctttt tagactggtc gtagggagac cacaacggtt
     2341 tccctctaga aataattttg tttaactata agaaggagat ataccatgcg gggttctcat
     2401 catcaccacc atcacagcag cggcctggtg ccgcgcggca gctcgctagc gctgccggtc
     2461 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     2521 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     2581 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     2641 cccaccctcg tgaccaccct gtcctggggc gtgcagtgct tcgcccgcta ccccgaccac
     2701 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     2761 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     2821 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     2881 gggcacaagc tggagtacaa ctactttagc gacaacgtct atatcaccgc cgacaagcag
     2941 aagaacggca tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cggcgtgcag
     3001 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     3061 aaccactacc tgagcaccca gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac
     3121 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     3181 aagtcgacca gatctctggc cgaggcggca gctaaggagg ccgcggcgaa ggaggcagcc
     3241 gcgaaagaag cagccgcaaa ggaagcggcc gccaaggctg ctgcaagatc tctcggatcg
     3301 ggtgctacta acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct
     3361 tcgctagcgc tggatcccgc cacaatgggc agcgtgaagc tgatccccag cctgaccacc
     3421 gtgatcctcg tgaagtccat gctgcggaag cggagcttcg gcaacccctt caagtataat
     3481 acggagaccc tgtaccccgc tgacggcggc ctggaaggcg catgtgacat ggccctgaag
     3541 ctcgtgggcg ggggccacct gaactgcagc cttgagacca catacagatc caagaaaccc
     3601 gctacgaacc tcaagatgcc cggtgtctac aacgtggacc acagactgga acgaatcaaa
     3661 gaggccgacg atgagaccta cgtcgagctg cacgaggtgg ctgtggccag atacgtgggc
     3721 ctgggtggtg gcggaggtac aggcgggagt gtgagcgagc tgattaagga gaacatgcca
     3781 atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc
     3841 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtcgtcga gggcggccct
     3901 ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcag aaccttcatc
     3961 aagcaccctc ctggcatccc cgacttcttt aagcagtcct tccctgaggg cttcacatgg
     4021 gagagagtca ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc
     4081 caggacggct gcctcatcta caacgtcaag gttagaggga tgaacttccc agccaacggc
     4141 cctgtgatgc agaagaaaac actcggctgg gaggccagta atggccaact gactgaagag
     4201 cagatcgcag aatttaaaga ggctttctcc ctatttgaca aggacgggga tgggacgata
     4261 acaaccaagg agctggggac ggtgatgcgg tctctggggc agaaccccac agaagcggag
     4321 ctgcaggaca tgatcaatga agtagatgcc gacggtgacg gcacattcga cttccctgag
     4381 ttcctgacga tgatggcaag aaaaatgagt tatagagtca ctgaagagga aattagagaa
     4441 gcgttccgcg tgtttgataa ggacggcaat ggctacatcg gcgcagcaga gcttcgccac
     4501 gtgatgacag accttggaga gaagttaaca gatgaggagg ttgatgaaat gatcagggta
     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 agcaaccagg
     5221 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     5281 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     5341 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     5401 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     5461 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     5521 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     5581 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     5641 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     5701 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     5761 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     5821 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     5881 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     5941 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     6001 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     6061 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     6121 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     6181 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     6241 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttc
//
