LOCUS       sequence-373341-        7226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of triple tagged CD86 with mCherry2, mEYFP(Q69K), and 
            mEGFP in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7226)
  AUTHORS   Foust DJ, Piston DW
  TITLE     Measuring G Protein Activation with Spectrally Resolved Fluorescence
            Fluctuation Spectroscopy
  JOURNAL   bioRxiv 2021.11.03.467169
REFERENCE   2  (bases 1 to 7226)
  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: "bioRxiv 
            2021.11.03.467169"
COMMENT     Sequence Label: pmCherry2-CD86-mEYFP(Q69K)-mEGFP
FEATURES             Location/Qualifiers
     source          1..7226
                     /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             757..1464
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(902..920)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             join(2422..2424,2425..2427,2428..3138)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3172..3174,3175..3177,3178..3891)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     polyA_signal    4014..4135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4051..4070)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4105..4124
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4142..4597)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4279..4300)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4491..4510
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4624..4728
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4730..5087
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4730..4921
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4753..4773)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4938..5073
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5122..5916
                     /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             5122..5916
                     /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(5176..5195)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5786..5805
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6104..6123)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6148..6195
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6524..7112
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7013..7032
                     /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 cgaggaggat
      781 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
      841 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
      901 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
      961 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     1021 aagctgtcct tccccgaggg cttcaattgg gagcgcgtga tgaacttcga ggacggcggc
     1081 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     1141 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agtgtcgtac catgggctgg
     1201 gaggcctcca ctgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     1261 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     1321 aagaagcccg tgcagctgcc cggcgcctac aacgtcgaca tcaagttgga catcctttcc
     1381 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     1441 ggcggcatgg acgagctgta caagtccgga atggcccctc tgaagattca agcttatttc
     1501 aatgagactg cagacctgcc atgccaattt gcaaactctc aaaaccaaag cctgagtgag
     1561 ctagtagtat tttggcagga ccaggaaaac ttggttctga atgaggtata cttaggcaaa
     1621 gagaaatttg acagtgttca ttccaagtat atgggccgca caagttttga ttcggacagt
     1681 tggaccctga gacttcacaa tcttcagatc aaggacaagg gcttgtatca atgtatcatc
     1741 catcacaaaa agcccacagg aatgattcgc atccaccaga tgaattctga actgtcagtg
     1801 cttgctaact tcagtcaacc tgaaatagta ccaatttcta atataacaga aaatgtgtac
     1861 ataaatttga cctgctcatc tatacacggt tacccagaac ctaagaagat gagtgttttg
     1921 ctaagaacca agaattcaac tatcgagtat gatggtatta tgcagaaatc tcaagataat
     1981 gtcacagaac tgtacgacgt ttccatcagc ttgtctgttt cattccctga tgttacgagc
     2041 aatatgacca tcttctgtat tctggaaact gacaagacgc ggcttttatc ttcacctttc
     2101 tctatagagc ttgaggaccc tcagcctccc ccagaccaca ttccttggat tacagctgta
     2161 cttccaacag ttattatatg tgtgatggtt ttctgtctaa ttctatggaa atggaagaag
     2221 aagaagcggc ctcgcaactc ttataaatgt ggaaccaaca caatggagag ggaagagagt
     2281 gaacagacca agaaaagaga aaaaatccat atacctgaaa gatctgatga aacccagcgt
     2341 gtttttaaaa gttcgaagac atcttcatgc gacaaaagtg atacatgttt tggaggcgga
     2401 ccggtgcccc agtgggaggg aatggtgagc aagggcgagg agctgttcac cggggtggtg
     2461 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     2521 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     2581 ctgcccgtgc cctggcccac cctcgtgacc accttcggct acggcctgaa gtgcttcgcc
     2641 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     2701 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     2761 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     2821 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     2881 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     2941 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     3001 gtgctgctgc ccgacaacca ctacctgagc taccagtcca agctgagcaa agaccccaac
     3061 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     3121 atggacgagc tgtacaagtt cgctgcgctg ctggcaaccc cggtcgctac tatggtgagc
     3181 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     3241 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     3301 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     3361 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     3421 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     3481 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3541 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3601 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     3661 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     3721 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3781 acccagtcca agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3841 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc
     3901 gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct ttaaaaaacc
     3961 tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt
     4021 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4081 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttaaggcg
     4141 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     4201 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     4261 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     4321 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     4381 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     4441 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     4501 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     4561 acacccgccg cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat
     4621 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     4681 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtcc tgaggcggaa
     4741 agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     4801 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     4861 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     4921 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     4981 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
     5041 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagat cgatcaagag
     5101 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     5161 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     5221 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     5281 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg
     5341 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     5401 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     5461 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     5521 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     5581 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     5641 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     5701 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     5761 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     5821 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     5881 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     5941 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     6001 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     6061 atctcatgct ggagttcttc gcccacccta gggggaggct aactgaaaca cggaaggaga
     6121 caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacggtg
     6181 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     6241 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     6301 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     6361 cctcaggtta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6421 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6481 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6541 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6601 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6661 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6721 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6781 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6841 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6901 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6961 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     7021 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     7081 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     7141 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7201 tggataaccg tattaccgcc atgcat
//
