LOCUS       sequence-452449-        4181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EGFP, for MuSIC barcode construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4181)
  AUTHORS   Lu X, Pritko DJ, Abravanel ME, Huggins JR, Ogunleye O, Biswas T, 
            Ashy KC, Woods SK, Livingston MWT, Blenner MA, Birtwistle MR
  TITLE     Genetically Encoded Fluorescence Barcodes Allow for Single-Cell 
            Analysis via Spectral Flow Cytometry.
  JOURNAL   ACS Synth Biol. 2025 May 16;14(5):1533-1548. doi: 
            10.1021/acssynbio.4c00807. Epub 2025 May 6.
  PUBMED    40326708
REFERENCE   2  (bases 1 to 4181)
  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: 
            "10.1021/acssynbio.4c00807"; journalName: "ACS Synth Biol"; date: 
            "2025-05-16- 16"; volume: "14"; issue: "5"; pages: "1533-1548"
COMMENT     Sequence Label: pR-EGFP
FEATURES             Location/Qualifiers
     source          1..4181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     416..435
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(734..753)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    778..825
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      884..1472
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1373..1392
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1653..1956
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1957..2160
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2111..2131
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2208..2217
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2214..2216,2217..2219,2220..2930)
                     /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(2259..2280)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2520..2539)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2867..2888
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3009..3130
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3046..3065)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3100..3119
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3137..3592)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3274..3295)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3486..3505
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(3636..3656)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3680..3890
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3702..3898
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3749..3884
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3933..4181,1..546)
                     /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             join(3933..4181,1..546)
                     /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(3987..4006)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
       61 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
      121 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
      181 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
      241 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
      301 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
      361 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
      421 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
      481 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
      541 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
      601 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
      661 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
      721 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
      781 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggttcgt
      841 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
      901 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
      961 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     1021 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     1081 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     1141 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     1201 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     1261 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     1321 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     1381 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     1441 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     1501 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     1561 tggataaccg tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta
     1621 gttcatagcc catatatgga gttccggcta gccgttacat aacttacggt aaatggcccg
     1681 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     1741 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1801 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     1861 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     1921 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     1981 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     2041 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     2101 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     2161 accgctgatc agcctcgtgc ttactggctt atcgaaatgg atccgccgcc accatggtga
     2221 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     2281 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     2341 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     2401 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     2461 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     2521 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     2581 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     2641 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     2701 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     2761 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2821 gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2881 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag ggctccatgt
     2941 cttcataaat gaagacgaat tcactgtatg gaattctgaa acataaaatg aatgcaattg
     3001 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     3061 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     3121 atgtatctta aggcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     3181 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     3241 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     3301 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     3361 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     3421 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     3481 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     3541 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcagtactt
     3601 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     3661 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3721 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3781 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     3841 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     3901 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3961 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     4021 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     4081 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     4141 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt t
//
