LOCUS       sequence-383616-        6271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Compare the intensity of the genetically encoded calcium sensor 
            (GECI) jRCaMP1b 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-jRCaMP1b
FEATURES             Location/Qualifiers
     source          1..6271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(214..233)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    258..305
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      634..1222
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1123..1142
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1370..1673
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1675..1877
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1828..1848
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2080..2102
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2164..2183
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2225..2343
                     /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             2418..2435
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2445..2462
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2481..2490
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2487..2489,2490..2492,2493..3203)
                     /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(2532..2553)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2793..2812)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3140..3161
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3324..3380
                     /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(4724..4743)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4789..4844
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4843..4862)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4932..4979
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        5005..5109
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5111..5468
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5111..5302
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5134..5154)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5319..5454
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(5503..6271,1..26)
                     /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(5503..6271,1..26)
                     /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(5557..5576)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6167..6186
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc
       61 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
      121 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
      181 ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga caataccgga
      241 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg ttgggtcgtt
      301 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
      361 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
      421 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta
      481 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
      541 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
      601 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
      661 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
      721 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
      781 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
      841 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
      901 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
      961 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     1021 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     1081 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     1141 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     1201 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     1261 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgaggccg gcctattaat
     1321 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     1381 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     1441 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     1501 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtccgcccc
     1561 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttac
     1621 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atgctgatgc
     1681 ggttttggca gtacaccaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     1741 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
     1801 aatgtcgtaa taaccccgcc ccgttgacgc aaatgggcgg taggcgtgta cggtgggagg
     1861 tctatataag cagacgtcgt ttagtgaacc gtcagatcac tagatgcttt attgcggtag
     1921 tttatcacag ttaaattgct aacgccagtc tcgaacttaa cgtgcagaag ttggtcgtga
     1981 ggcactgggc aggtaagtat cgggcccttt gtgcgggggg agcggctcgg ggctgtccgc
     2041 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
     2101 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
     2161 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg gatcggaccg
     2221 aaatcaccac aattcagcaa attgtgaaca tcatcacgtt catctttccc tggttgccaa
     2281 aggcccattt tcctgtcagt aacgagaagg tcgcgaattc aggcgctttt tagactggtc
     2341 gtagggagac cacaacggtt tccctctaga aataattttg tttaactata agaaggagat
     2401 ataccatgcg gggttctcat catcaccacc atcacagcag cggcctggtg ccgcgcggca
     2461 gctcgctagc gctgccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg
     2521 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2581 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2641 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gtcctggggc gtgcagtgct
     2701 tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2761 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2821 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2881 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctactttagc gacaacgtct
     2941 atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc cgccacaaca
     3001 tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3061 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
     3121 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3181 tcggcatgga cgagctgtac aagtcgacca gatctctggc cgaggcggca gctaaggagg
     3241 ccgcggcgaa ggaggcagcc gcgaaagaag cagccgcaaa ggaagcggcc gccaaggctg
     3301 ctgcaagatc tctcggatcg ggtgctacta acttcagcct gctgaagcag gctggagacg
     3361 tggaggagaa ccctggacct tcgctagcgc tggatcccgc cacaatggtc gactcatcgc
     3421 gacgtaagtg gaataagtgg ggtcacgcag tcagagctat aggtcggctg agctcagcga
     3481 acaacaccga aatgatgtac ccagcggatg gtggtctgcg tggttacact cacatggcgc
     3541 tgaaagttga tggcggcggt cacctgtcct gttctttcgt gaccacctac cgctccaaaa
     3601 agactgtcgg caacattaag atgcctgcca ttcattacgt cagccaccgt ctggagcgcc
     3661 tggaggagag cgataacgaa atgtttgtcg tacagcgtga acacgcagtt gccaagtttg
     3721 tgggcctggg tggtggcggc ggtaccggag ggagcatgaa ctccctgatc aaggagaaca
     3781 tgcgtatgaa agtggttctg gaaggctccg taaacggcca ccagttcaaa tgcactggtg
     3841 aaggcgaagg caacccgtat atgggcaccc agactatgcg tatcaaagtg atcgagggtg
     3901 gtccgctgcc gtttgcgttc gacatcctgg cgacgtcctt tatgtatggc tcccgtacct
     3961 tcatcaaata tccgaaaggc atcccggatt tctttaagca gtccttcccg gaaggtttta
     4021 cctgggaacg tgtgacccgt tacgaagacg gcggcgtaat taccgttatg caagacacgt
     4081 ctctggagga tggctgcctg gtgtatcacg tgcaggttcg cggtgtgaac ttcccgagca
     4141 atggtgctgt aatgcaaaag aaaaccaaag gttgggagcc tacggactcc caactgactg
     4201 aagagcagat cgcagaattt aaagaggctt tctccctatt tgacaaggac ggggatggga
     4261 caataacaac caaggagatg gggacggtga tgcggtctct ggggcagaac cccacagaag
     4321 cagagctgca ggacatgatc aatgaagtag atgccgacgg tgacggcaca atcgacttcc
     4381 ctgagttcct gattatgatg gcaggcaaaa tgaaatacac agacagtgaa gaagaaatta
     4441 gagaagcgtt cggcgtgttt gataaggatg gcaatggcta catcagtgca gcagagcttc
     4501 gccacgtgat gacaaacctt ggagagaagt taacagatga agaggttgat gaaatgatca
     4561 gggaagcaga cagcgatggg gatggtcagg taaactacga agagtttgta caaatgatga
     4621 cagcgaagta gaagcttgtg attaacctca ggtgcaggct gcctatcaga aggtggtggc
     4681 tggtgtggcc aatgccctgg ctcacaaata ccactgagat cgatcttttt ccctctgcca
     4741 aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat
     4801 ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg
     4861 ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg
     4921 cccatatgta actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     4981 tgaaagcatg ctcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     5041 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5101 aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa
     5161 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     5221 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     5281 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     5341 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     5401 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     5461 tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga
     5521 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa
     5581 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt
     5641 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg
     5701 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa
     5761 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac
     5821 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt
     5881 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact
     5941 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg
     6001 ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg
     6061 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc
     6121 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt
     6181 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc
     6241 gccgctcccg attcgcagcg catcgccttc t
//
