LOCUS       sequence-272542-        5086 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CD9-pHluorin for visualization of multivesicular 
            body-plasma membrane fusion..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5086)
  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 5086)
  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-CD9-pHluorin
FEATURES             Location/Qualifiers
     source          1..5086
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(23..40)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(194..782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(274..293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(860..893)
                     /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(995..1786,1787..1855))
                     /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     1618..1637
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     enhancer        2226..2529
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2530..2733
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2684..2704
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2730..2754
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     2859..2875
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2859..2875
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        2886..2904
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2886..2903
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    2905..2929
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      3108..3117
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3114..3830
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(3147..3175)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3770..3790
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    complement(4429..4453)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(4454..4473)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4455..4473)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4484..4501)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4484..4500)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4493..4515)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     polyA_signal    4905..5039
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4942..4961)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4996..5015
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
ORIGIN
        1 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
       61 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      121 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      181 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      241 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      301 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      361 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      421 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      481 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      541 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      601 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      661 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      721 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      781 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      841 aagggatttt ggtcatgcca taacttcgta tagcatacat tatacgaagt tatggcatga
      901 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
      961 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     1021 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     1081 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     1141 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     1201 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     1261 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     1321 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     1381 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     1441 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     1501 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     1561 cattctgaga atagtgtatg cggcgaccga gttgctcttg tccggcgtca atacgggata
     1621 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     1681 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     1741 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     1801 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     1861 tcctttttca atattattga agcatttatc agggttattg tctcatgcca ggggtgggca
     1921 cacatatttg ataccagcga tccctacaca gcacataatt caatgcgact tccctctatc
     1981 gcacatctta gacctttatt ctccctccag cacacatcga agctgccgag caagccgttc
     2041 tcaccagtcc aagacctggc atgagcggat acatatttga atgtatttag aaaaataaac
     2101 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     2161 ttatcatgac attaacctat aaaaataggc gtattacgag gccctttcac gcattagatg
     2221 catgtcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
     2281 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
     2341 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
     2401 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
     2461 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
     2521 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
     2581 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
     2641 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
     2701 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
     2761 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
     2821 actctagcct aggccgcggg acggataaca atttcacaca ggaaacagct atgaccatta
     2881 ggcctattta ggtgacacta tagaacaagt ttgtacaaaa aagcaggctg gtaccggtcc
     2941 ggaattccat gccggtcaaa ggaggcacca agtgcatcaa atacctgctg ttcggattta
     3001 acttcatctt ctggcttgcc gggattgctg tccttgccat tggactatgg ctccgattcg
     3061 actctcagac caagagcatc ttcgagcaag aaactaatag atctctagcc accatgggaa
     3121 gtaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta gatggtgatg
     3181 ttaatgggca caaattttct gtcagtggag agggtgaagg tgatgcaaca tacggaaaac
     3241 ttacccttaa atttatttgc actactggaa aactacctgt tccttggcca acacttgtca
     3301 ctactttaac ttatggtgtt caatgctttt caagataccc agatcatatg aaacggcatg
     3361 actttttcaa gagtgccatg cccgaaggtt atgtacagga aagaactata tttttcaaag
     3421 atgacgggaa ctacaagaca cgtgctgaag tcaagtttga aggtgatacc cttgttaata
     3481 gaatcgagtt aaaaggtatt gattttaaag aagatggaaa cattcttgga cacaaattgg
     3541 aatacaacta taacgatcac caggtgtaca tcatggcaga caaacaaaag aatggaatca
     3601 aagctaactt caaaattaga cacaacattg aagatggagg cgttcaacta gcagaccatt
     3661 atcaacaaaa tactccaatt ggcgatgggc ccgtcctttt accagacaac cattacctgt
     3721 ttacaacttc tactctttcg aaagatccca acgaaaagag agaccacatg gtccttcttg
     3781 agtttgtaac agctgctggg attacacatg gcatggatga actatacaaa accggtggag
     3841 ctcgaatcag atctaataat aattccagct tctacacagg agtctatatt ctgatcggag
     3901 ccggcgccct catgatgctg gtgggcttcc tgggctgctg cggggctgtg caggagtccc
     3961 agtgcatgct gggactgttc ttcggcttcc tcttggtgat attcgccatt gaaatagctg
     4021 cggccatctg gggatattcc cacaaggatg aggtgattaa ggaagtccag gagttttaca
     4081 aggacaccta caacaagctg aaaaccaagg atgagcccca gcgggaaacg ctgaaagcca
     4141 tccactatgc gttgaactgc tgtggtttgg ctgggggcgt ggaacagttt atctcagaca
     4201 tctgccccaa gaaggacgta ctcgaaacct tcaccgtgaa gtcctgtcct gatgccatca
     4261 aagaggtctt cgacaataaa ttccacatca tcggcgcagt gggcatcggc attgccgtgg
     4321 tcatgatatt tggcatgatc ttcagtatga tcttgtgctg tgctatccgc aggaaccgcg
     4381 agatggtcta ggtctagagt atccctcgag gggcccaagc ttacgcgtac ccagctttct
     4441 tgtacaaagt ggtccctata gtgagtcgta ttataagcta ggcactggcc gtcgttttac
     4501 aacgtcgtga ctgggaaaac tgctagcttg ggatctttgt gaaggaacct tacttctgtg
     4561 gtgtgacata attggacaaa ctacctacag agatttaaag ctctaaggta aatataaaat
     4621 ttttaagtgt ataatgtgtt aaactagctg catatgcttg ctgcttgaga gttttgctta
     4681 ctgagtatga tttatgaaaa tattatacac aggagctagt gattctaatt gtttgtgtat
     4741 tttagattca cagtcccaag gctcatttca ggcccctcag tcctcacagt ctgttcatga
     4801 tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac ctcccacacc
     4861 tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg tttattgcag
     4921 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4981 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg
     5041 atcctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgc
//
