LOCUS       sequence-373335-        6482 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CD86 double tagged with mEYFP(Q69K) and mEGFP in 
            mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6482)
  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 6482)
  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: pCD86-mEYFP(Q69K)-mEGFP
FEATURES             Location/Qualifiers
     source          1..6482
                     /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"
     CDS             join(1678..1680,1681..1683,1684..2394)
                     /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(2428..2430,2431..2433,2434..3147)
                     /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    3270..3391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3307..3326)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3361..3380
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3398..3853)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3535..3556)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3747..3766
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3880..3984
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3986..4343
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3986..4177
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4009..4029)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4194..4329
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4378..5172
                     /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             4378..5172
                     /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(4432..4451)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5042..5061
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5360..5379)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5404..5451
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5780..6368
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6269..6288
                     /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 gattcaagct tatttcaatg agactgcaga cctgccatgc
      781 caatttgcaa actctcaaaa ccaaagcctg agtgagctag tagtattttg gcaggaccag
      841 gaaaacttgg ttctgaatga ggtatactta ggcaaagaga aatttgacag tgttcattcc
      901 aagtatatgg gccgcacaag ttttgattcg gacagttgga ccctgagact tcacaatctt
      961 cagatcaagg acaagggctt gtatcaatgt atcatccatc acaaaaagcc cacaggaatg
     1021 attcgcatcc accagatgaa ttctgaactg tcagtgcttg ctaacttcag tcaacctgaa
     1081 atagtaccaa tttctaatat aacagaaaat gtgtacataa atttgacctg ctcatctata
     1141 cacggttacc cagaacctaa gaagatgagt gttttgctaa gaaccaagaa ttcaactatc
     1201 gagtatgatg gtattatgca gaaatctcaa gataatgtca cagaactgta cgacgtttcc
     1261 atcagcttgt ctgtttcatt ccctgatgtt acgagcaata tgaccatctt ctgtattctg
     1321 gaaactgaca agacgcggct tttatcttca cctttctcta tagagcttga ggaccctcag
     1381 cctcccccag accacattcc ttggattaca gctgtacttc caacagttat tatatgtgtg
     1441 atggttttct gtctaattct atggaaatgg aagaagaaga agcggcctcg caactcttat
     1501 aaatgtggaa ccaacacaat ggagagggaa gagagtgaac agaccaagaa aagagaaaaa
     1561 atccatatac ctgaaagatc tgatgaaacc cagcgtgttt ttaaaagttc gaagacatct
     1621 tcatgcgaca aaagtgatac atgttttgga ggcggaccgg tgccccagtg ggagggaatg
     1681 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc
     1741 gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc
     1801 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc
     1861 gtgaccacct tcggctacgg cctgaagtgc ttcgcccgct accccgacca catgaagcag
     1921 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc
     1981 aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg
     2041 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag
     2101 ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc
     2161 atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac
     2221 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac
     2281 ctgagctacc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg
     2341 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagttcgct
     2401 gcgctgctgg caaccccggt cgctactatg gtgagcaagg gcgaggagct gttcaccggg
     2461 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     2521 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     2581 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     2641 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2701 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2761 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2821 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     2881 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     2941 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     3001 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccaagct gagcaaagac
     3061 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     3121 ctcggcatgg acgagctgta caagtaaagc ggccgcgact ctagatcata atcagccata
     3181 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     3241 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     3301 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3361 gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg
     3421 ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc
     3481 ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt
     3541 tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc
     3601 tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg
     3661 tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga
     3721 aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg
     3781 ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg
     3841 ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     3901 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     3961 caataatatt gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc
     4021 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     4081 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     4141 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     4201 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     4261 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     4321 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     4381 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     4441 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     4501 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     4561 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     4621 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     4681 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     4741 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     4801 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     4861 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     4921 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     4981 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     5041 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     5101 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     5161 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     5221 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     5281 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     5341 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     5401 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg
     5461 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     5521 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     5581 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt
     5641 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     5701 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     5761 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     5821 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     5881 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     5941 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6001 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6061 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6121 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6181 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6241 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6301 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     6361 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     6421 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc
     6481 at
//
