LOCUS       sequence-383661-        5331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mEGFP-mCherry in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5331)
  AUTHORS   Obashi K, Sochacki KA, Strub MP, Taraska JW
  TITLE     A conformational switch in clathrin light chain regulates lattice 
            structure and endocytosis at the plasma membrane of mammalian cells.
  JOURNAL   Nat Commun. 2023 Feb 9;14(1):732. doi: 10.1038/s41467-023-36304-7.
  PUBMED    36759616
REFERENCE   2  (bases 1 to 5331)
  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: "Nat 
            Commun."; date: "2023-02-9"; volume: "14(1):732. doi"; pages: " 
            10.1038/s41467-023-36304-7"
COMMENT     Sequence Label: EGFP-mCherry
FEATURES             Location/Qualifiers
     source          1..5331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        74..377
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        378..581
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     532..552
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      620..629
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(626..628,629..631,632..1144)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(671..692)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(932..951)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1279..1300
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1458..1476)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1726..1745
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2132..2253
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2169..2188)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2223..2242
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2260..2715)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2397..2418)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2609..2628
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2742..2846
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2848..3205
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2848..3039
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2871..2891)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3056..3191
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3240..4034
                     /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             3240..4034
                     /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(3294..3313)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3904..3923
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4222..4241)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4266..4313
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4642..5230
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5131..5150
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taccgccatg cattagttat taatagtaat caattacggg gtcattagtt catagcccat
       61 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      121 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      181 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      241 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      301 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      361 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      421 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      481 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      541 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tggtttagtg aaccgtcaga
      601 tccgctagcg ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt
      661 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      721 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      781 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
      841 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      901 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
      961 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     1021 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     1081 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     1141 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1201 ccccgtgctg ctgcccgaca accactacct gagcacccag tccaagctga gcaaagaccc
     1261 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg gcggatccgg
     1321 agggtctgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     1381 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     1441 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     1501 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     1561 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     1621 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     1681 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     1741 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     1801 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     1861 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     1921 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     1981 gggccgccac tccaccggcg gcatggacga gctgtacaag taactgatca taatcagcca
     2041 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     2101 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     2161 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2221 ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt
     2281 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     2341 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     2401 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     2461 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     2521 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2581 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2641 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2701 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2761 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2821 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     2881 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2941 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3001 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3061 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3121 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     3181 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     3241 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3301 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3361 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3421 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3481 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3541 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     3601 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     3661 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     3721 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     3781 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3841 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3901 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     3961 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4021 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4081 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4141 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4201 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     4261 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     4321 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     4381 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     4441 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     4501 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4561 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4621 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4681 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4741 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4801 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4861 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4921 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4981 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5041 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5101 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5161 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5221 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5281 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta t
//
