LOCUS       sequence-259045-        5080 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CD63-pHuji for visualization of multivesicular 
            body-plasma membrane fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5080)
  AUTHORS   Verweij FJ, Bebelman MP, Jimenez CR, Garcia-Vallejo JJ, Janssen H, 
            Neefjes J, Knol JC, de Goeij-de Haas R, Piersma SR, Baglio SR, 
            Verhage M, Middeldorp JM, Zomer A, van Rheenen J, Coppolino MG, 
            Hurbain I, Raposo G, Smit MJ, Toonen RFG, van Niel G, Pegtel DM
  TITLE     Quantifying exosome secretion from single cells reveals a modulatory
            role for GPCR signaling.
  JOURNAL   J Cell Biol. 2018 Mar 5;217(3):1129-1142. doi: 
            10.1083/jcb.201703206. Epub 2018 Jan 16.
  PUBMED    29339438
REFERENCE   2  (bases 1 to 5080)
  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"; doi: 
            "10.1083/jcb.201703206"; journalName: "J Cell Biol"; date: 
            "2018-03-5- 5"; volume: "217"; issue: "3"; pages: "1129-1142"
COMMENT     Sequence Label: pCMV-Sport6-CD63-pHuji
FEATURES             Location/Qualifiers
     source          1..5080
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(5..27)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     polyA_signal    417..551
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(454..473)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     508..527
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(621..638)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(792..1380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(872..891)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(1458..1491)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1593..2384,2385..2453))
                     /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     2216..2235
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     enhancer        2824..3127
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3128..3331
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3282..3302
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     3328..3352
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     3457..3473
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3457..3473
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3484..3502
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3484..3501
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    3503..3527
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3661..4368
                     /codon_start=1
                     /product="pH-sensitive red fluorescent protein derived from
                     mApple, for imaging of exocytosis and endocytosis (Shen et 
                     al., 2014)"
                     /label=pHuji
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNF
                     EDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALK
                     SEIKYRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     4074..4093
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     protein_bind    complement(5021..5045)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(5046..5065)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5047..5065)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(join(5076..5080,1..13))
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(join(5076..5080,1..12))
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 ccgtcgtttt acaacgtcgt gactgggaaa actgctagct tgggatcttt gtgaaggaac
       61 cttacttctg tggtgtgaca taattggaca aactacctac agagatttaa agctctaagg
      121 taaatataaa atttttaagt gtataatgtg ttaaactagc tgcatatgct tgctgcttga
      181 gagttttgct tactgagtat gatttatgaa aatattatac acaggagcta gtgattctaa
      241 ttgtttgtgt attttagatt cacagtccca aggctcattt caggcccctc agtcctcaca
      301 gtctgttcat gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
      361 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
      421 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
      481 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
      541 atgtctggat cgatcctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
      601 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
      661 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
      721 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
      781 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
      841 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
      901 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
      961 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     1021 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     1081 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     1141 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     1201 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     1261 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     1321 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     1381 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     1441 gggattttgg tcatgccata acttcgtata gcatacatta tacgaagtta tggcatgaga
     1501 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     1561 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     1621 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     1681 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     1741 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     1801 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     1861 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     1921 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     1981 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     2041 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     2101 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     2161 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgtc cggcgtcaat acgggataat
     2221 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     2281 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     2341 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     2401 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     2461 ctttttcaat attattgaag catttatcag ggttattgtc tcatgccagg ggtgggcaca
     2521 catatttgat accagcgatc cctacacagc acataattca atgcgacttc cctctatcgc
     2581 acatcttaga cctttattct ccctccagca cacatcgaag ctgccgagca agccgttctc
     2641 accagtccaa gacctggcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     2701 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     2761 atcatgacat taacctataa aaataggcgt attacgaggc cctttcacgc attagatgca
     2821 tgtcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     2881 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     2941 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     3001 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     3061 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     3121 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     3181 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     3241 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     3301 tgtacggtgg gaggtctata taagcagagc tcgtttagtg aaccgtcaga tcgcctggag
     3361 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac
     3421 tctagcctag gccgcgggac ggataacaat ttcacacagg aaacagctat gaccattagg
     3481 cctatttagg tgacactata gaacaagttt gtacaaaaaa gcaggctggt accggtccgg
     3541 aattccatgg cggtggaagg aggaatgaaa tgtgtgaagt tcttgctcta cgtcctcctg
     3601 ctggcctttt gcgcctgtgc agtgggactg attgccgtgg gtgtcggggc acagagatct
     3661 atggtgagca agggcgagga gaataacatg gccatcatca aggagttcat gcgcttcaag
     3721 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc
     3781 cgcccctacg aggcctttca gaccgctaag ctgaaggtga ccaagggtgg ccccctgccc
     3841 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggtcta cattaagcac
     3901 ccagccgaca tccccgacta cttcaagctg tccttccccg agggcttcag gtgggagcgc
     3961 gtgatgaact tcgaggacgg cggcattatt cacgttaacc aggactcctc cctgcaggac
     4021 ggcgtgttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta
     4081 atgcagaaga agaccatggg ctgggaggcc tccgaggagc ggatgtaccc cgaggacggc
     4141 gccctgaaga gcgagattaa gtataggctg aagctgaagg acggcggcca ctacgccgcc
     4201 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacatcgtg
     4261 gacatcaagt tggacatcgt gtcccacaac gaggactaca ccatcgtgga acagtacgaa
     4321 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagag atctcttgtc
     4381 ctgagtcaga ccataatcca gggggctacc cctggctctc tgttgccagt ggtcatcatc
     4441 gcagtgggtg tcttcctctt cctggtggct tttgtgggct gctgcggggc ctgcaaggag
     4501 aactattgtc ttatgatcac gtttgccatc tttctgtctc ttatcatgtt ggtggaggtg
     4561 gccgcagcca ttgctggcta tgtgtttaga gataaggtga tgtcagagtt taataacaac
     4621 ttccggcagc agatggagaa ttacccgaaa aacaaccaca ctgcttcgat cctggacagg
     4681 atgcaggcag attttaagtg ctgtggggct gctaactaca cagattggga gaaaatccct
     4741 tccatgtcga agaaccgagt ccccgactcc tgctgcatta atgttactgt gggctgtggg
     4801 attaatttca acgagaaggc gatccataag gagggctgtg tggagaagat tgggggctgg
     4861 ctgaggaaaa atgtgctggt ggtagctgca gcagcccttg gaattgcttt tgtcgaggtt
     4921 ttgggaattg tctttgcctg ctgcctcgtg aagagtatca gaagtggcta cgaggtgatg
     4981 taggtctaga gtatccctcg aggggcccaa gcttacgcgt acccagcttt cttgtacaaa
     5041 gtggtcccta tagtgagtcg tattataagc taggcactgg
//
