LOCUS       sequence-374750-        6509 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of CD86 double tagged with mCherry2 (N-term) and mEGFP 
            (C-term) in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6509)
  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 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"; journalName: "bioRxiv 
            2021.11.03.467169"
COMMENT     Sequence Label: pmCherry2-CD86-mEGFP
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             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(2455..2457,2458..2460,2461..3174)
                     /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"
     primer_bind     complement(2500..2521)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2761..2780)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3108..3129
                     /label=EGFP-C
                     /note="EGFP, 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 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 attcgctgcg ctgctggcaa ccccggtcgc tactatggtg
     2461 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     2521 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     2581 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     2641 accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac
     2701 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     2761 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     2821 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     2881 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     2941 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     3001 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     3061 agcacccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     3121 gagttcgtga ccgccgccgg gatcactctc 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
//
