LOCUS       sequence-325828-        6941 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV promoter-LoxP-mKate-LoxP-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6941)
  AUTHORS   Somiya M, Kuroda S
  TITLE     Reporter gene assay for membrane fusion of extracellular vesicles.
  JOURNAL   J Extracell Vesicles. 2021 Nov;10(13):e12171. doi: 
            10.1002/jev2.12171.
  PUBMED    34807503
REFERENCE   2  (bases 1 to 6941)
  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: "J Extracell
            Vesicles."; date: "2021-11"; volume: "10(13):e12171. doi"; pages: " 
            10.1002/jev2.12171"
COMMENT     Sequence Label: pcDNA-LoxP-mKate-LoxP-EGFP
FEATURES             Location/Qualifiers
     source          1..6941
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        115..494
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        495..698
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     649..669
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     743..762
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        743..761
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(772..805)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      821..830
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             827..1540
                     /codon_start=1
                     /product="monomeric derivative of red fluorescent protein 
                     from Entacmaea quadricolor (Merzlyak et al., 2007)"
                     /label=TagRFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRSD
                     MALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     protein_bind    complement(1550..1583)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1618..1627
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1624..1626,1627..1629,1630..2343)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1624..1626,1627..1629,1630..2340)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1669..1690)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1930..1949)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2277..2298
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2374..2392)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2375..2392)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2412..2429)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2418..2642
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2688..3116
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2775..2794)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2985..3006
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3125..3145)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3130..3459
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3310..3445
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3372..3391
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3526..4320
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3580..3599)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4190..4209
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4494..4615
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4531..4550)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4585..4604
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4664..4680)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4664..4680)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4677..4699)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4688..4704
                     /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(4712..4718,4719..4736,4737..4742))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4757..4778
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4895..4912)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5066..5654)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5146..5165)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5825..6616,6617..6685))
                     /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     6448..6467
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6686..6790)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6865..6884)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
       61 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      121 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      181 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      241 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      301 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      361 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      421 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      481 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      541 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      601 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      661 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      721 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gataacttcg
      781 tatagcatac attatacgaa gttataattc tgcacatagt gccaccatgg tgtctaaggg
      841 cgaagagctg attaaggaga acatgcacat gaagctgtac atggagggca ccgtgaacaa
      901 ccaccacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat
      961 gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag
     1021 cttcatgtac ggcagcaaaa ccttcatcaa ccacacccag ggcatccccg acttctttaa
     1081 gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt
     1141 gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat
     1201 cagaggggtg aacttcccat ccaacggccc tgtgatgcag aagaaaacac tcggctggga
     1261 ggcctccacc gagatgctgt accccgctga cggcggcctg gaaggcagaa gcgacatggc
     1321 cctgaagctc gtgggcgggg gccacctgat ctgcaacttg aagaccacat acagatccaa
     1381 gaaacccgct aagaacctca agatgcccgg cgtctactat gtggacagaa gactggaaag
     1441 aatcaaggag gccgacaaag agacctacgt cgagcagcac gaggtggctg tggccagata
     1501 ctgcgacctc cctagcaaac tggggcacaa acttaattag ttctagttga taacttcgta
     1561 tagcatacat tatacgaagt tattcgagtc tagagggccc gttagatcga acgcgtcgcc
     1621 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1681 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1741 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1801 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     1861 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1921 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1981 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2041 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2101 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2161 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2221 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2281 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2341 taatagctcg agcatgcatc tagagggccc tattctatag tgtcacctaa atgctagagc
     2401 tcgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2461 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2521 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2581 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2641 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag
     2701 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     2761 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2821 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2881 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2941 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     3001 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     3061 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt
     3121 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt
     3181 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     3241 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     3301 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     3361 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     3421 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     3481 tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
     3541 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3601 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     3661 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     3721 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     3781 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     3841 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     3901 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     3961 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4021 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     4081 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4141 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4201 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4261 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4321 gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt
     4381 tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg
     4441 gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt
     4501 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4561 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4621 tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     4681 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     4741 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     4801 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     4861 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     4921 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     4981 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5041 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     5101 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     5161 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     5221 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     5281 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacaaaccc cccgttcagc
     5341 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     5401 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     5461 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     5521 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     5581 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     5641 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     5701 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     5761 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     5821 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     5881 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     5941 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6001 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6061 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6121 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6181 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6241 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6301 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     6361 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6421 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6481 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6541 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6601 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6661 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6721 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     6781 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg ggagatctcc
     6841 cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta
     6901 tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc g
//
