LOCUS       sequence-262710-        6509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of CD86 N-terminally tagged with EGFP and 
            C-terminally tagged with mApple.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6509)
  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 6509)
  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-CD86-mApple
FEATURES             Location/Qualifiers
     source          1..6509
                     /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"
     misc_feature    591..671
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      673..682
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      751..760
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(757..759,760..762,763..1473)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(757..759,760..762,763..1473)
                     /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(802..823)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1063..1082)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1410..1431
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2464..3174
                     /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     2877..2896
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3297..3418
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3334..3353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3388..3407
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3425..3880)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3562..3583)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3774..3793
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3907..4011
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4013..4370
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4013..4204
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4036..4056)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4221..4356
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4405..5199
                     /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             4405..5199
                     /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(4459..4478)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5069..5088
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5387..5406)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5431..5478
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5807..6395
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6296..6315
                     /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 caagcttcga attctgcagt cgacggtacc gcgggcccgg
      661 gatccaccgg tcgccaccat gggactgagt aacattctct ttgtgatggc cttcctgctc
      721 tctggtgctg ctcctctgaa gattccggtc gccaccatgg tgagcaaggg cgaggagctg
      781 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
      841 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
      901 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
      961 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     1021 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     1081 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     1141 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     1201 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     1261 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     1321 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg
     1381 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
     1441 gggatcactc tcggcatgga cgagctgtac aagtccggaa tggcccctct gaagattcaa
     1501 gcttatttca atgagactgc agacctgcca tgccaatttg caaactctca aaaccaaagc
     1561 ctgagtgagc tagtagtatt ttggcaggac caggaaaact tggttctgaa tgaggtatac
     1621 ttaggcaaag agaaatttga cagtgttcat tccaagtata tgggccgcac aagttttgat
     1681 tcggacagtt ggaccctgag acttcacaat cttcagatca aggacaaggg cttgtatcaa
     1741 tgtatcatcc atcacaaaaa gcccacagga atgattcgca tccaccagat gaattctgaa
     1801 ctgtcagtgc ttgctaactt cagtcaacct gaaatagtac caatttctaa tataacagaa
     1861 aatgtgtaca taaatttgac ctgctcatct atacacggtt acccagaacc taagaagatg
     1921 agtgttttgc taagaaccaa gaattcaact atcgagtatg atggtattat gcagaaatct
     1981 caagataatg tcacagaact gtacgacgtt tccatcagct tgtctgtttc attccctgat
     2041 gttacgagca atatgaccat cttctgtatt ctggaaactg acaagacgcg gcttttatct
     2101 tcacctttct ctatagagct tgaggaccct cagcctcccc cagaccacat tccttggatt
     2161 acagctgtac ttccaacagt tattatatgt gtgatggttt tctgtctaat tctatggaaa
     2221 tggaagaaga agaagcggcc tcgcaactct tataaatgtg gaaccaacac aatggagagg
     2281 gaagagagtg aacagaccaa gaaaagagaa aaaatccata tacctgaaag atctgatgaa
     2341 acccagcgtg tttttaaaag ttcgaagaca tcttcatgcg acaaaagtga tacatgtttt
     2401 ggaggcggac cggtgcccca gtgggaggga ttcgctgcgc tgctggcaac cccggtcgct
     2461 actatggtga gcaagggcga ggagaataac atggccatca tcaaggagtt catgcgcttc
     2521 aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     2581 ggccgcccct acgaggcctt tcagaccgct aagctgaagg tgaccaaggg tggccccctg
     2641 cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggt ctacattaag
     2701 cacccagccg acatccccga ctacttcaag ctgtccttcc ccgagggctt caggtgggag
     2761 cgcgtgatga acttcgagga cggcggcatt attcacgtta accaggactc ctccctgcag
     2821 gacggcgtgt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     2881 gtaatgcaga agaagaccat gggctgggag gcctccgagg agcggatgta ccccgaggac
     2941 ggcgccctga agagcgagat caagaagagg ctgaagctga aggacggcgg ccactacgcc
     3001 gccgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacatc
     3061 gtcgacatca agttggacat cgtgtcccac aacgaggact acaccatcgt ggaacagtac
     3121 gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gtaaagcggc
     3181 cgcgactcta gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
     3241 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
     3301 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3361 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaag
     3421 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     3481 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     3541 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     3601 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     3661 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     3721 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     3781 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3841 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     3901 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3961 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg
     4021 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     4081 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     4141 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     4201 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     4261 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     4321 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa
     4381 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4441 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4501 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4561 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     4621 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4681 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4741 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4801 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4861 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4921 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4981 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     5041 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     5101 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     5161 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     5221 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     5281 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     5341 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg
     5401 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
     5461 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     5521 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     5581 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     5641 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     5701 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     5761 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     5821 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     5881 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     5941 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6001 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6061 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6121 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6181 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6241 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6301 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6361 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6421 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     6481 ctgtggataa ccgtattacc gccatgcat
//
