LOCUS       sequence-262735-        6037 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HRas N-terminally tagged with mApple and 
            EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6037)
  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 6037)
  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: pmApple-EGFP-HRas
FEATURES             Location/Qualifiers
     source          1..6037
                     /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"
     CDS             628..1335
                     /codon_start=1
                     /product="photostable monomeric derivative of DsRed (Shaner
                     et al., 2008)"
                     /label=mApple
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEA
                     FQTAKLKVTKGGPLPFAWDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNF
                     EDGGIIHVNQDSSLQDGVFIYKVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGALK
                     SEIKKRLKLKDGGHYAAEVKTTYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     1041..1060
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     regulatory      1384..1393
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1390..1392,1393..1395,1396..2106)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1390..1392,1393..1395,1396..2106)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1435..1456)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1696..1715)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2043..2064
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2825..2946
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2862..2881)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2916..2935
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2953..3408)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3090..3111)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3302..3321
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3435..3539
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3541..3898
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3541..3732
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3564..3584)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3749..3884
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3933..4727
                     /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             3933..4727
                     /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(3987..4006)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4597..4616
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4915..4934)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4959..5006
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5335..5923
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5824..5843
                     /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 ccggactcag atctcgagct caagcttatg gtgagcaagg gcgaggagaa taacatggcc
      661 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
      721 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg cctttcagac cgctaagctg
      781 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
      841 tacggctcca aggtctacat taagcaccca gccgacatcc ccgactactt caagctgtcc
      901 ttccccgagg gcttcaggtg ggagcgcgtg atgaacttcg aggacggcgg cattattcac
      961 gttaaccagg actcctccct gcaggacggc gtgttcatct acaaggtgaa gctgcgcggc
     1021 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     1081 gaggagcgga tgtaccccga ggacggcgcc ctgaagagcg agatcaagaa gaggctgaag
     1141 ctgaaggacg gcggccacta cgccgccgag gtcaagacca cctacaaggc caagaagccc
     1201 gtgcagctgc ccggcgccta catcgtcgac atcaagttgg acatcgtgtc ccacaacgag
     1261 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     1321 gacgagctgt acaagtccgg actcagatct cgagcggtac cgcgggcccg ggatccaccg
     1381 gtcgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc
     1441 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat
     1501 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     1561 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     1621 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc
     1681 accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     1741 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     1801 ctggggcaca agctggagta caactacaac agccacaacg tctatatcat ggccgacaag
     1861 cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg
     1921 cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc
     1981 gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat
     2041 cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg
     2101 tacaagtact cagatctcat gaccgaatac aagcttgttg ttgttggcgc cggcggtgtg
     2161 ggcaagagtg cgctgaccat ccagctgatc cagaaccatt ttgtggacga atacgacccc
     2221 actatagagg attcctaccg gaagcaggtg gtcattgatg gggagacgtg cctgttggac
     2281 atcctggata ccgccggcca ggaggagtac agcgccatgc gggaccagta catgcgcacc
     2341 ggggagggct tcctgtgtgt gtttgccatc aacaacacca agtcttttga ggacatccac
     2401 cagtacaggg agcagatcaa acgggtgaag gactcggatg acgtgcccat ggtgctggtg
     2461 gggaacaagt gtgacctggc tgcacgcact gtggaatctc ggcaggctca ggacctcgcc
     2521 cgaagctacg gcatccccta catcgagacc tcggccaaga cccggcaggg agtggaggat
     2581 gccttctaca cgttggtgcg tgagatccgg cagcacaagc tgcggaagct gaaccctcct
     2641 gatgagagtg gccccggctg catgagctgc aagtgtgtgc tctcctgagg gcccgggatc
     2701 caccggatct agataactga tcataatcag ccataccaca tttgtagagg ttttacttgc
     2761 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     2821 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2881 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2941 atcttaacgc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     3001 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     3061 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     3121 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     3181 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     3241 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     3301 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     3361 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     3421 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3481 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     3541 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     3601 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3661 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3721 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3781 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     3841 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     3901 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3961 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     4021 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     4081 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     4141 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     4201 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     4261 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     4321 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     4381 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     4441 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     4501 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     4561 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     4621 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4681 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4741 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4801 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4861 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     4921 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     4981 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     5041 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     5101 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     5161 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     5221 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     5281 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     5341 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     5401 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     5461 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     5521 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     5581 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     5641 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5701 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5761 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5821 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5881 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5941 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     6001 ccctgattct gtggataacc gtattaccgc catgcat
//
