LOCUS       sequence-452469-        4181 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mClover3, 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-mClover3
FEATURES             Location/Qualifiers
     source          1..4181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(25..735,736..738,739..741))
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(67..88)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     675..696
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      738..747
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(795..998)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(824..844)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        999..1302
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(1483..2071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1563..1582)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2130..2177
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     2202..2221
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(2409..3203)
                     /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(2409..3203)
                     /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(2520..2539)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3130..3149
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3238..3434)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        complement(3246..3456)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3252..3387
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3480..3500
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3544..3999
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3631..3650)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3841..3862
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4006..4127
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4017..4036)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     4071..4090
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
ORIGIN
        1 cttcaattat gaagacatgg agcccttgta cagctcgtcc atgccatgtg taatcccggc
       61 ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag
      121 cttggactga tggctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg
      181 ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcaacgt tgtggcggat
      241 cttgaagtta gccttgatgc agttcttctg cttgtcggcc gtgatataga cgtagtggct
      301 gttgaagttg tactccagct tgtgccccag gatgttgccg tcctccttga agtcgatgcc
      361 cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt
      421 gtaggtaccg tcgtccttga aagagatggt gcgctcctgg acgtagcctt cgggcatggc
      481 ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc aggccacgcc
      541 gtagccgaag gtggtcacga gggtgggcca gggcacgggc agcttgccgg tggtgcagat
      601 gaacttcagg gtcagcttgc cgttggtggc atcgccctcg ccctcgccgc ggacgctgaa
      661 cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc cggtgaacag
      721 ctcctcgccc ttgctcacca tggtggcggc ggatccattt cgataagcca gtaagcacga
      781 ggctgatcag cggtagctct gcttatatag acctcccacc gtacacgcct accgcccatt
      841 tgcgtcaatg gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa
      901 aacaaactcc cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc
      961 tatccacgcc cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat
     1021 acgtagatgt actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag
     1081 gcgggccatt taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga
     1141 tgtactgcca agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc
     1201 cctattggcg ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg
     1261 ggcggtcagc caggcgggcc atttaccgta agttatgtaa cggctagccg gaactccata
     1321 tatgggctat gaactaatga ccccgtaatt gattactatt aataactaat gcatggcggt
     1381 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     1441 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     1501 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     1561 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     1621 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     1681 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     1741 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     1801 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     1861 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     1921 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     1981 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     2041 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     2101 tgacgctcag tggaacgaac cgcgtttatg aacaaacgac ccaacaccgt gcgttttatt
     2161 ctgtcttttt attgccgtca tagcgcgggt tccttccggt attgtctcct tccgtgtttc
     2221 agttagcctc cccctagggt gggcgaagaa ctccagcatg agatccccgc gctggaggat
     2281 catccagccg gcgtcccgga aaacgattcc gaagcccaac ctttcataga aggcggcggt
     2341 ggaatcgaaa tctcgtgatg gcaggttggg cgtcgcttgg tcggtcattt cgaaccccag
     2401 agtcccgctc agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga
     2461 gcggcgatac cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca
     2521 atatcacggg tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag
     2581 tcgatgaatc cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca
     2641 tgggtcacga cgagatcctc gccgtcgggc atgctcgcct tgagcctggc gaacagttcg
     2701 gctggcgcga gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc
     2761 atccgagtac gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc
     2821 ggatcaagcg tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga
     2881 gcaaggtgag atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt
     2941 cccgcttcag tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac
     3001 gatagccgcg ctgcctcgtc ttgcagttca ttcagggcac cggacaggtc ggtcttgaca
     3061 aaaagaaccg ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt
     3121 gtctgttgtg cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg
     3181 tgcaatccat cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcgatcttt
     3241 gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct cagaggccga
     3301 ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg gagaatgggc
     3361 ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta tggttgctga
     3421 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     3481 ccctaactga cacacattcc acagctggtt ctttccgcct caggactctt cctttaagta
     3541 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3601 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3661 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3721 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     3781 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     3841 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3901 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3961 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta cgccttaaga tacattgatg
     4021 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
     4081 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
     4141 gcattcattt tatgtttcag aattccgaca tctgaattcg t
//
