LOCUS       sequence-413387-        6782 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tandem mutant calcium-buffer with rapalog dimerizer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6782)
  AUTHORS   Davis LC, Morgan AJ, Galione A
  TITLE     NAADP-regulated two-pore channels drive phagocytosis through 
            endo-lysosomal Ca(2+) nanodomains, calcineurin and dynamin.
  JOURNAL   EMBO J. 2020 Jul 15;39(14):e104058. doi: 10.15252/embj.2019104058. 
            Epub 2020 Jun 8.
  PUBMED    32510172
REFERENCE   2  (bases 1 to 6782)
  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: "EMBO J."; 
            date: "2020-07-15"; volume: "39(14):e104058. doi"; pages: " 
            10.15252/embj.2019104058. Epub 2020 Jun 8"
COMMENT     Sequence Label: FKBP12-CB2mutx2-mTagBFP2-HAx2
FEATURES             Location/Qualifiers
     source          1..6782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(622..624,625..945)
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNK
                     PFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVEL
                     LKLE"
     CDS             2692..3390
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3391..3417
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3421..3447
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3570..3691
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3607..3626)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3661..3680
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3698..4153)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3835..3856)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4047..4066
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4180..4284
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4286..4643
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4286..4477
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4309..4329)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4494..4629
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4678..5472
                     /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             4678..5472
                     /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(4732..4751)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5342..5361
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5660..5679)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5704..5751
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6080..6668
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6569..6588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggactcag atctcgagac catgggagtg caggtggaaa ccatctcccc aggagacggg
      661 cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact acaccgggat gcttgaagat
      721 ggaaagaaat ttgattcctc ccgggacaga aacaagccct ttaagtttat gctaggcaag
      781 caggaggtga tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg tcagagagcc
      841 aaactgacta tatctccaga ttatgcctat ggtgccactg ggcacccagg catcatccca
      901 ccacatgcca ctctcgtctt cgatgtggag cttctaaaac tggaagctcg aggcgctcga
      961 ggcgcagcag cgggagcagg aggagcagct agcatggctg gcccgcagca gcagccccct
     1021 tacctgcacc tggccgagct gacggcgtcc cagttcctgg aaatatggaa gcactttgac
     1081 gcagacggaa atgggtatat tgaaggtaaa cagctagaaa actttttcca agagctggag
     1141 aaggcaagga aaggctctgg catgatgtca aagagtgaca actttggaga aaagatgaag
     1201 gagttcatgc agaagtatga taaaaactca gatgggaaaa tcgagatggc acagctggcg
     1261 cagatcctgc caaccgaaga gaacttcctt ctgtgcttca ggcagcacgt gggctccagc
     1321 accgagttta tggaggcttg gcggaagtac gacacagaca ggagtggcta catcgaagcc
     1381 aatcagctca agggattcct gtcagacctg ctgaagaagg cgaaccggcc gtacgatgag
     1441 cccaagctcc aggaatacac ccaaaccata ctacggatgt ttgacttgaa cggggatggc
     1501 aaattgggcc tctcacagat gtcccgactc ctgcctgtcc aggaaaactt cctgcttaaa
     1561 tttcagggca tgaagctgac ctcagaggag tttaacgcga tcttcacatt ttacgacaag
     1621 gatagaagcg gctacattga cgagcatcag ctggatgccc ttttgaagga tctgtacgag
     1681 aaaaacaaaa aggaaatgaa tattcaacag ctcaccaact acagaaagag cgtcatgtcc
     1741 ttggcagagg cagggaagct ctaccgcaag gacctggaga ttgtgctctg cagcgagccc
     1801 cccatggggg atcctaaacc ggtgatcgga gcaggaggag cagctagcat ggctggcccg
     1861 cagcagcagc ccccttacct gcacctggcc gagctgacgg cgtcccagtt cctggaaata
     1921 tggaagcact ttgacgcaga cggaaatggg tatattgaag gtaaacagct agaaaacttt
     1981 ttccaagagc tggagaaggc aaggaaaggc tctggcatga tgtcaaagag tgacaacttt
     2041 ggagaaaaga tgaaggagtt catgcagaag tatgataaaa actcagatgg gaaaatcgag
     2101 atggcacagc tggcgcagat cctgccaacc gaagagaact tccttctgtg cttcaggcag
     2161 cacgtgggct ccagcaccga gtttatggag gcttggcgga agtacgacac agacaggagt
     2221 ggctacatcg aagccaatca gctcaaggga ttcctgtcag acctgctgaa gaaggcgaac
     2281 cggccgtacg atgagcccaa gctccaggaa tacacccaaa ccatactacg gatgtttgac
     2341 ttgaacgggg atggcaaatt gggcctctca cagatgtccc gactcctgcc tgtccaggaa
     2401 aacttcctgc ttaaatttca gggcatgaag ctgacctcag aggagtttaa cgcgatcttc
     2461 acattttacg acaaggatag aagcggctac attgacgagc atcagctgga tgcccttttg
     2521 aaggatctgt acgagaaaaa caaaaaggaa atgaatattc aacagctcac caactacaga
     2581 aagagcgtca tgtccttggc agaggcaggg aagctctacc gcaaggacct ggagattgtg
     2641 ctctgcagcg agccccccat gggggatcct aaaccggtac cggtcgccac catgagcgag
     2701 ctgattaagg agaacatgca catgaagctg tacatggagg gcaccgtgga caaccatcac
     2761 ttcaagtgca catccgaggg cgaaggcaag ccctacgagg gcacccagac catgagaatc
     2821 aaggtggtcg agggcggccc tctccccttc gccttcgaca tcctggctac tagcttcctc
     2881 tacggcagca agaccttcat caaccacacc cagggcatcc ccgacttctt caagcagtcc
     2941 ttccctgagg gcttcacatg ggagagagtc accacatacg aagacggggg cgtgctgacc
     3001 gctacccagg acaccagcct ccaggacggc tgcctcatct acaacgtcaa gatcagaggg
     3061 gtgaacttca catccaacgg ccctgtgatg cagaagaaaa cactcggctg ggaggccttc
     3121 accgagacgc tgtaccccgc tgacggcggc ctggaaggca gaaacgacat ggccctgaag
     3181 ctcgtgggcg ggagccatct gatcgcaaac gccaagacca catatagatc caagaaaccc
     3241 gctaagaacc tcaagatgcc tggcgtctac tatgtggact acagactgga aagaatcaag
     3301 gaggccaaca acgagaccta cgtcgagcag cacgaggtgg cagtggccag atactgcgac
     3361 ctccctagca aactggggca caagcttaat tacccttacg atgtgcctga ctacgccccc
     3421 tacccctacg acgtgcccga ttatgcttaa ggccgcgact ctagatcata atcagccata
     3481 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     3541 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     3601 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3661 gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg
     3721 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     3781 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     3841 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     3901 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     3961 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     4021 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     4081 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     4141 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4201 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4261 caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc
     4321 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4381 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4441 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     4501 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4561 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     4621 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     4681 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     4741 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4801 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     4861 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4921 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4981 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     5041 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     5101 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     5161 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     5221 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     5281 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     5341 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     5401 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     5461 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     5521 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     5581 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     5641 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     5701 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg
     5761 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     5821 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     5881 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt
     5941 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6001 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6061 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6121 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6181 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6241 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6301 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6361 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6421 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6481 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6541 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6601 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6661 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6721 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc
     6781 at
//
