LOCUS       sequence-371440-        7523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses BRET Ca2+ sensor CalfluxCTN in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7523)
  AUTHORS   Cumberbatch D, Mori T, Yang J, Mi D, Vinson P, Weaver CD, Johnson CH
  TITLE     A BRET Ca(2+) sensor enables high-throughput screening in the 
            presence of background fluorescence.
  JOURNAL   Sci Signal. 2022 Aug 16;15(747):eabq7618. doi: 
            10.1126/scisignal.abq7618. Epub 2022 Aug 16.
  PUBMED    35973028
REFERENCE   2  (bases 1 to 7523)
  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: "Sci 
            Signal."; date: "2022-08-16"; volume: "15(747):eabq7618. doi"; 
            pages: " 10.1126/scisignal.abq7618. Epub 2022 Aug 16"
COMMENT     Sequence Label: pCAG-CalfluxCTN
FEATURES             Location/Qualifiers
     source          1..7523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          656..1672
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1596..1618
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1680..1699
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1831..1840
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1882..1903)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2490..2511
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2770..3285
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(3379..3396)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3385..3609
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3655..4083
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3742..3761)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3952..3973
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4092..4112)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4097..4426
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4277..4412
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4339..4358
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4437..5036
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4437..4456)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4933..4953
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5196..5317
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5233..5252)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5287..5306
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5366..5382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5366..5382)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5379..5401)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5390..5406
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5414..5420,5421..5438,5439..5444))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5459..5480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5597..5614)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5768..6356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5848..5867)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6527..7318,7319..7387))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7150..7169
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7388..7492)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggs gsgcgccrgg cggggsgggg sggggsgrgg ggsggggcgg
      541 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt
      601 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt
      661 cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg
      721 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg
      781 ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct
      841 tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg
      901 tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg
      961 gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg
     1021 tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt
     1081 gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc
     1141 tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc
     1201 ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc
     1261 gcctcgggcc ggggrgggct cgggggrggg gcgcggcggc ccccggagcg ccggcggctg
     1321 tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg
     1381 acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct
     1501 tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg
     1561 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     1621 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg
     1681 caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattcctcg aggaattcac
     1741 tcctcaggtg caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca
     1801 caaataccac tgagatctgc atgagctagc gccaccatgg tgagcaaggg cgaggagctg
     1861 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     1921 agcgtccgcg gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc
     1981 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc
     2041 gtggcctgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2101 atgcccgaag gctacgtcca ggagcgcacc atctctttca aggacgacgg tacctacaag
     2161 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2221 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc
     2281 cacaacgtct atatcacggc cgacaagcag aagaacggca tcaaggctaa cttcaagatc
     2341 cgccacaacg ttgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     2401 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagccatca gtccgccctg
     2461 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     2521 gggattatgc aagttgccga cgccagcgaa gaggaactga gcgagtgctt cagaatcttc
     2581 gacttcgacg gcaacggctt catcgacaga gaggagtttg gcgacatcat cagactgacc
     2641 ggcgagcagc tgaccgacga ggacgtggac gagatcttcg gcgactccga caccgacaag
     2701 aacggcagaa tcgatttcga cgagttcctg aagatggtgg aaaacgtgca gcccatctac
     2761 cccgagctca tggtcttcac actcgaagat ttcgttgggg actggcgaca gacagccggc
     2821 tacaacctgg accaagtcct tgaacaggga ggtgtgtcca gtttgtttca gaatctcggg
     2881 gtgtccgtaa ctccgatcca aaggattgtc ctgagcggtg aaaatgggct gaagatcgac
     2941 atccatgtca tcatcccgta tgaaggtctg agcggcgacc aaatgggcca gatcgaaaaa
     3001 atttttaagg tggtgtaccc tgtggatgat catcacttta aggtgatcct gcactatggc
     3061 acactggtaa tcgacggggt tacgccgaac atgatcgact atttcggacg gccgtatgaa
     3121 ggcatcgccg tgttcgacgg caaaaagatc actgtaacag ggaccctttg gaacggcaac
     3181 aaaattatcg acgagcgcct gatcaacccc gacggctccc tgctgttccg agtaaccatc
     3241 aacggagtga ccggctggcg gctgtgcgaa cgcattctgg cgtaaggatc cactagtcca
     3301 gtgtggtgga attctgcaga tatccagcac agtggcggcc gctcgagtct agagggcccg
     3361 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     3421 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3481 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3541 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3601 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     3661 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3721 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3781 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3841 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3901 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3961 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     4021 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     4081 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     4141 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     4201 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4261 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4321 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     4381 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgcatga
     4441 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     4501 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     4561 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     4621 gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg
     4681 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     4741 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     4801 cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg
     4861 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     4921 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     4981 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgattcg
     5041 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     5101 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5161 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5221 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5281 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5341 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5401 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5461 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5521 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5581 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5641 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5701 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5761 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5821 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5881 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5941 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     6001 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6061 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6121 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6181 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     6241 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6301 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6361 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6421 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6481 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6541 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6601 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6661 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6721 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6781 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6841 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6901 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6961 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     7021 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7081 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7141 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7201 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7261 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7321 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7381 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7441 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7501 cccgaaaagt gccacctgac gtc
//
