LOCUS       sequence-454231-        4181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EBFP2, 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-EBFP2
FEATURES             Location/Qualifiers
     source          1..4181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(64..858)
                     /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             complement(64..858)
                     /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(175..194)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     785..804
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(893..1089)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(901..1111)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      907..1042
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1135..1155
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1199..1654
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1286..1305)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1496..1517
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1661..1782
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1672..1691)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1726..1745
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(1861..2571,2572..2574,2575..2577))
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     2511..2532
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2574..2583
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2631..2834)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2660..2680)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2835..3138
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3319..3907)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3399..3418)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3966..4013
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4038..4057
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
ORIGIN
        1 cgaaatctcg tgatggcagg ttgggcgtcg cttggtcggt catttcgaac cccagagtcc
       61 cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc
      121 gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc
      181 acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat
      241 gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt
      301 cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg
      361 cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg
      421 agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc
      481 aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag
      541 gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc
      601 ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag
      661 ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag
      721 aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg
      781 ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa
      841 tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcga tctttgcaaa
      901 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg
      961 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     1021 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     1081 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacccta
     1141 actgacacac attccacagc tggttctttc cgcctcagga ctcttccttt aagtactgac
     1201 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     1261 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     1321 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     1381 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     1441 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     1501 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     1561 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     1621 gcgaatttta acaaaatatt aacgcttaca atttacgcct taagatacat tgatgagttt
     1681 ggacaaacca caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     1741 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     1801 cattttatgt ttcagaattc attatctaga attcgtcttc aattatgaag acatggagcc
     1861 cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtgcggaact ccagcaggac
     1921 catgtgatcg cgcttctcgt tggggtcttt gctcagcacg gactgggtgc tcaggtagtg
     1981 gctgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc
     2041 gagctgcacg ctgccgtcct ccacgttgtg gcggatcttg aagttcacct tgatgccgtt
     2101 cttctgcttg acggccatga tatagatgtt gtggctgttg aagttgtact ccagcttgtg
     2161 ccccaggatg ttgccgtcct ccttgaagtc gacgcccttc agctcgatgc ggttcactag
     2221 ggtgtcgccc tcgaacttca cctcggcgcg ggtcttgtag gtgccgtcgt ccttgaagaa
     2281 gatggtgcgc tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt
     2341 catgtggtcg gggtagcggg cgaagcactg cacgccgtgg ctcagggtgg tcacgagggt
     2401 gggccagggc acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgtt
     2461 ggtggcatcg ccctcgccct cgcccctcac gctgaacttg tggccgttta cgtcgccgtc
     2521 cagctcgacc aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatggt
     2581 ggcggcggat ccatttcgat aagccagtaa gcacgaggct gatcagcggt agctctgctt
     2641 atatagacct cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta
     2701 cgacattttg gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg
     2761 gggtggagac ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc
     2821 caaaaccgca tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga
     2881 aagtcccata aggtcatgta ctgggcataa tgccaggcgg gccatttacc gtcattgacg
     2941 tcaatagggg gcgtacttgg catatgatac acttgatgta ctgccaagtg ggcagtttac
     3001 cgtaaatact ccacccattg acgtcaatgg aaagtcccta ttggcgttac tatgggaaca
     3061 tacgtcatta ttgacgtcaa tgggcggggg tcgttgggcg gtcagccagg cgggccattt
     3121 accgtaagtt atgtaacggc tagccggaac tccatatatg ggctatgaac taatgacccc
     3181 gtaattgatt actattaata actaatgcat ggcggtaata cggttatcca cagaatcagg
     3241 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     3301 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     3361 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     3421 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     3481 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     3541 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     3601 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     3661 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     3721 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     3781 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     3841 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     3901 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaccgcg
     3961 tttatgaaca aacgacccaa caccgtgcgt tttattctgt ctttttattg ccgtcatagc
     4021 gcgggttcct tccggtattg tctccttccg tgtttcagtt agcctccccc tagggtgggc
     4081 gaagaactcc agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac
     4141 gattccgaag cccaaccttt catagaaggc ggcggtggaa t
//
