LOCUS       sequence-330217-        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        127..506
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        507..710
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     661..681
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     755..774
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        755..773
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(784..817)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      833..842
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             839..1552
                     /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(1562..1595)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      1630..1639
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1636..1638,1639..1641,1642..2355)
                     /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(1636..1638,1639..1641,1642..2352)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1681..1702)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1942..1961)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2289..2310
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        complement(2386..2404)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2387..2404)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2424..2441)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2430..2654
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2700..3128
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2787..2806)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2997..3018
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3137..3157)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3142..3471
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3322..3457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3384..3403
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3538..4332
                     /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(3592..3611)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4202..4221
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4506..4627
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4543..4562)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4597..4616
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4676..4692)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4676..4692)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4689..4711)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4700..4716
                     /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(4724..4730,4731..4748,4749..4754))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4769..4790
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4907..4924)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5078..5666)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5158..5177)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5837..6628,6629..6697))
                     /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     6460..6479
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6698..6802)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6877..6896)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
       61 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      121 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      181 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      241 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      301 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      361 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      421 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      481 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      541 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      601 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      661 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
      721 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
      781 aagataactt cgtatagcat acattatacg aagttataat tctgcacata gtgccaccat
      841 ggtgtctaag ggcgaagagc tgattaagga gaacatgcac atgaagctgt acatggaggg
      901 caccgtgaac aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
      961 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     1021 cctggctacc agcttcatgt acggcagcaa aaccttcatc aaccacaccc agggcatccc
     1081 cgacttcttt aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     1141 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     1201 caacgtcaag atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac
     1261 actcggctgg gaggcctcca ccgagatgct gtaccccgct gacggcggcc tggaaggcag
     1321 aagcgacatg gccctgaagc tcgtgggcgg gggccacctg atctgcaact tgaagaccac
     1381 atacagatcc aagaaacccg ctaagaacct caagatgccc ggcgtctact atgtggacag
     1441 aagactggaa agaatcaagg aggccgacaa agagacctac gtcgagcagc acgaggtggc
     1501 tgtggccaga tactgcgacc tccctagcaa actggggcac aaacttaatt agttctagtt
     1561 gataacttcg tatagcatac attatacgaa gttattcgag tctagagggc ccgttagatc
     1621 gaacgcgtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     1681 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     1741 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     1801 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     1861 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     1921 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1981 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     2041 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     2101 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     2161 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     2221 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     2281 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     2341 gagctgtaca agtaatagct cgagcatgca tctagagggc cctattctat agtgtcacct
     2401 aaatgctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2461 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2521 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2581 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2641 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     2701 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     2761 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     2821 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     2881 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     2941 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3001 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     3061 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3121 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     3181 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     3241 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     3301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     3361 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     3421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     3481 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     3541 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     3601 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     3661 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     3721 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     3781 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     3841 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     3901 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     3961 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4021 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     4081 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4141 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     4201 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     4261 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     4321 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     4381 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     4441 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     4501 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4561 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4621 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     4681 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4741 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4801 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     4861 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     4921 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     4981 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5041 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5101 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5161 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5221 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5281 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacaaac
     5341 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5401 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5461 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5521 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5581 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5641 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     5701 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5761 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     5821 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     5881 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     5941 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6001 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6061 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6121 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6181 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6241 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6301 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6361 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6421 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6481 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6541 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6601 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6661 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     6721 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     6781 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     6841 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     6901 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc g
//
