LOCUS       sequence-413478-        6758 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tandem calcium-buffer with rapalog dimerizer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6758)
  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 6758)
  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-CB2x2-mTagBFP2-HAx2
FEATURES             Location/Qualifiers
     source          1..6758
                     /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             2668..3366
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             3367..3393
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3397..3423
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3546..3667
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3583..3602)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3637..3656
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3674..4129)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3811..3832)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4023..4042
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4156..4260
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4262..4619
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4262..4453
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4285..4305)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4470..4605
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4654..5448
                     /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             4654..5448
                     /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(4708..4727)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5318..5337
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5636..5655)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5680..5727
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6056..6644
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6545..6564
                     /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 aggcgcagca
      961 gcgggagcag gaggagcagc tagcatggct ggcccgcagc agcagccccc ttacctgcac
     1021 ctggccgagc tgacggcgtc ccagttcctg gaaatatgga agcactttga cgcagacgga
     1081 aatgggtata ttgaaggtaa agagctagaa aactttttcc aagagctgga gaaggcaagg
     1141 aaaggctctg gcatgatgtc aaagagtgac aactttggag aaaagatgaa ggagttcatg
     1201 cagaagtatg ataaaaactc agatgggaaa atcgagatgg cagagctggc gcagatcctg
     1261 ccaaccgaag agaacttcct tctgtgcttc aggcagcacg tgggctccag caccgagttt
     1321 atggaggctt ggcggaagta cgacacagac aggagtggct acatcgaagc caatgagctc
     1381 aagggattcc tgtcagacct gctgaagaag gcgaaccggc cgtacgatga gcccaagctc
     1441 caggaataca cccaaaccat actacggatg tttgacttga acggggatgg caaattgggc
     1501 ctctcagaga tgtcccgact cctgcctgtc caggaaaact tcctgcttaa atttcagggc
     1561 atgaagctga cctcagagga gtttaacgcg atcttcacat tttacgacaa ggatagaagc
     1621 ggctacattg acgagcatga gctggatgcc cttttgaagg atctgtacga gaaaaacaaa
     1681 aaggaaatga atattcaaca gctcaccaac tacagaaaga gcgtcatgtc cttggcagag
     1741 gcagggaagc tctaccgcaa ggacctggag attgtgctct gcagcgagcc ccccatgggg
     1801 gatccaccgg tcgccaccgt cgacggtacc accatggctg gcccgcagca gcagccccct
     1861 tacctgcacc tggccgagct gacggcgtcc cagttcctgg aaatatggaa gcactttgac
     1921 gcagacggaa atgggtatat tgaaggtaaa gagctagaaa actttttcca agagctggag
     1981 aaggcaagga aaggctctgg catgatgtca aagagtgaca actttggaga aaagatgaag
     2041 gagttcatgc agaagtatga taaaaactca gatgggaaaa tcgagatggc agagctggcg
     2101 cagatcctgc caaccgaaga gaacttcctt ctgtgcttca ggcagcacgt gggctccagc
     2161 accgagttta tggaggcttg gcggaagtac gacacagaca ggagtggcta catcgaagcc
     2221 aatgagctca agggattcct gtcagacctg ctgaagaagg cgaaccggcc gtacgatgag
     2281 cccaagctcc aggaatacac ccaaaccata ctacggatgt ttgacttgaa cggggatggc
     2341 aaattgggcc tctcagagat gtcccgactc ctgcctgtcc aggaaaactt cctgcttaaa
     2401 tttcagggca tgaagctgac ctcagaggag tttaacgcga tcttcacatt ttacgacaag
     2461 gatagaagcg gctacattga cgagcatgag ctggatgccc ttttgaagga tctgtacgag
     2521 aaaaacaaaa aggaaatgaa tattcaacag ctcaccaact acagaaagag cgtcatgtcc
     2581 ttggcagagg cagggaagct ctaccgcaag gacctggaga ttgtgctctg cagcgagccc
     2641 cccatggggg atccaccggt cgccaccatg agcgagctga ttaaggagaa catgcacatg
     2701 aagctgtaca tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa
     2761 ggcaagccct acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc
     2821 cccttcgcct tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac
     2881 cacacccagg gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag
     2941 agagtcacca catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag
     3001 gacggctgcc tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct
     3061 gtgatgcaga agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac
     3121 ggcggcctgg aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc
     3181 gcaaacgcca agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc
     3241 gtctactatg tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc
     3301 gagcagcacg aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag
     3361 cttaattacc cttacgatgt gcctgactac gccccctacc cctacgacgt gcccgattat
     3421 gcttaaggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     3481 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     3541 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3601 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3661 tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     3721 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     3781 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     3841 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     3901 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     3961 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     4021 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     4081 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     4141 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4201 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4261 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     4321 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     4381 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4441 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4501 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4561 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     4621 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     4681 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4741 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     4801 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     4861 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4921 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4981 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     5041 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     5101 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     5161 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     5221 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     5281 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     5341 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     5401 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     5461 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     5521 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     5581 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     5641 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     5701 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     5761 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     5821 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     5881 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     5941 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6001 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6061 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6121 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6181 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6241 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6301 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6361 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6421 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6481 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6541 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6601 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6661 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6721 cccctgattc tgtggataac cgtattaccg ccatgcat
//
