LOCUS       sequence-262719-        5932 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HRas N-terminally tagged with EGFP and 
            mApple.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5932)
  AUTHORS   Foust DJ, Godin AG, Ustione A, Wiseman PW, Piston DW
  TITLE     Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions
            between Membrane Proteins.
  JOURNAL   Biophys J. 2019 Nov 5;117(9):1764-1777. doi: 
            10.1016/j.bpj.2019.09.028. Epub 2019 Sep 28.
  PUBMED    31606123
REFERENCE   2  (bases 1 to 5932)
  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: 
            "10.1016/j.bpj.2019.09.028"; journalName: "Biophys J"; date: 
            "2019-11-5- 5"; volume: "117"; issue: "9"; pages: "1764-1777"
COMMENT     Sequence Label: pEGFP(1-233)-mApple(2-236)-HRas(2-189)
FEATURES             Location/Qualifiers
     source          1..5932
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1131)
                     /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(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1312..2016
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="VSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAF
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNFE
                     DGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALKS
                     EIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     1722..1741
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    2720..2841
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2757..2776)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2811..2830
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2848..3303)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2985..3006)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3197..3216
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3330..3434
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3436..3793
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3436..3627
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3459..3479)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3644..3779
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3828..4622
                     /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             3828..4622
                     /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(3882..3901)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4492..4511
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4810..4829)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4854..4901
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5230..5818
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5719..5738
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg cgtgagcaag
     1321 ggcgaggaga ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     1381 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     1441 gcctttcaga ccgctaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     1501 atcctgtccc ctcagttcat gtacggctcc aaggtctaca ttaagcaccc agccgacatc
     1561 cccgactact tcaagctgtc cttccccgag ggcttcaggt gggagcgcgt gatgaacttc
     1621 gaggacggcg gcattattca cgttaaccag gactcctccc tgcaggacgg cgtgttcatc
     1681 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaag
     1741 accatgggct gggaggcctc cgaggagcgg atgtaccccg aggacggcgc cctgaagagc
     1801 gagatcaaga agaggctgaa gctgaaggac ggcggccact acgccgccga ggtcaagacc
     1861 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acatcgtcga catcaagttg
     1921 gacatcgtgt cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     1981 cgccactcca ccggcggcat ggacgagctg tacaagaccg aatacaagct tgttgttgtt
     2041 ggcgccggcg gtgtgggcaa gagtgcgctg accatccagc tgatccagaa ccattttgtg
     2101 gacgaatacg accccactat agaggattcc taccggaagc aggtggtcat tgatggggag
     2161 acgtgcctgt tggacatcct ggataccgcc ggccaggagg agtacagcgc catgcgggac
     2221 cagtacatgc gcaccgggga gggcttcctg tgtgtgtttg ccatcaacaa caccaagtct
     2281 tttgaggaca tccaccagta cagggagcag atcaaacggg tgaaggactc ggatgacgtg
     2341 cccatggtgc tggtggggaa caagtgtgac ctggctgcac gcactgtgga atctcggcag
     2401 gctcaggacc tcgcccgaag ctacggcatc ccctacatcg agacctcggc caagacccgg
     2461 cagggagtgg aggatgcctt ctacacgttg gtgcgtgaga tccggcagca caagctgcgg
     2521 aagctgaacc ctcctgatga gagtggcccc ggctgcatga gctgcaagtg tgtgctctcc
     2581 tgagggcccg ggatccaccg gatctagata actgatcata atcagccata ccacatttgt
     2641 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     2701 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     2761 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     2821 caaactcatc aatgtatctt aacgcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     2881 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2941 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     3001 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     3061 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     3121 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     3181 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     3241 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     3301 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3361 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3421 gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     3481 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3541 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3601 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3661 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3721 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3781 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3841 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3901 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3961 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     4021 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4081 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4141 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4201 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4261 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4321 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     4381 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4441 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4501 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4561 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4621 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4681 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4741 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     4801 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     4861 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     4921 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     4981 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     5041 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     5101 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5161 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5221 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     5281 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5341 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5401 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5461 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5521 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5581 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5641 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5701 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5761 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5821 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5881 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc at
//
