LOCUS       sequence-373333-        5765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mEGFP-tagged CD86 in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5765)
  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 5765)
  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-mEGFP
FEATURES             Location/Qualifiers
     source          1..5765
                     /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(1711..1713,1714..1716,1717..2430)
                     /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(1756..1777)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2017..2036)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2364..2385
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2553..2674
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2590..2609)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2644..2663
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2681..3136)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2818..2839)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3030..3049
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3163..3267
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3269..3626
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3269..3460
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3292..3312)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3477..3612
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3661..4455
                     /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             3661..4455
                     /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(3715..3734)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4325..4344
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4643..4662)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4687..4734
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5063..5651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5552..5571
                     /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 ggagggattc
     1681 gctgcgctgc tggcaacccc ggtcgctact atggtgagca agggcgagga gctgttcacc
     1741 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1801 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1861 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     1921 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     1981 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2041 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2101 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     2161 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     2221 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     2281 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa gctgagcaaa
     2341 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     2401 actctcggca tggacgagct gtacaagtaa agcggccgcg actctagatc ataatcagcc
     2461 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc
     2521 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt
     2581 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2641 gttgtggttt gtccaaactc atcaatgtat cttaaggcgt aaattgtaag cgttaatatt
     2701 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2761 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2821 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2881 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2941 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     3001 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     3061 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     3121 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     3181 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     3241 cttcaataat attgaaaaag gaagagtcct gaggcggaaa gaaccagctg tggaatgtgt
     3301 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3361 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta
     3421 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     3481 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     3541 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     3601 tttttggagg cctaggcttt tgcaaagatc gatcaagaga caggatgagg atcgtttcgc
     3661 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     3721 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     3781 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     3841 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     3901 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     3961 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4021 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4081 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4141 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     4201 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4261 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4321 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4381 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4441 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4501 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4561 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     4621 cccaccctag ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     4681 ctatgacggc aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac
     4741 gcggggttcg gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc
     4801 aatacgcccg cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag
     4861 ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac
     4921 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4981 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5041 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5101 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5161 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5221 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5281 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5341 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5401 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5461 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5521 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5581 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5641 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5701 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcca
     5761 tgcat
//
