LOCUS       sequence-452492-        3767 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mRuby3 and extra insert, for MuSIC barcode 
            construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3767)
  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 3767)
  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: pM-mRuby3
FEATURES             Location/Qualifiers
     source          1..3767
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    417..538
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      592..1180
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1081..1100
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1355..1658
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1659..1862
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1813..1833
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1889..1945
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1955..2008
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      2018..2027
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2024..2734
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTD
                     IALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAV
                     AKYSNLGGGMDELYK"
     polyA_signal    2772..2893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2900..3355)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3037..3058)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3249..3268
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3381..3483
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(3484..3767,1..376)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3550..3569)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca
       61 gtttctacac atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc
      121 taaagggttt attgagaata tgtttttcgt atcagccaat ccctgggtga gtttcaccag
      181 ttttgattta aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa
      241 atattatacg caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt
      301 ttgtgatggc ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg
      361 gcagggcggg gcgtaatcaa acatgagaat tggtcgcgcg agcaattcag acatgataag
      421 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
      481 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttgg
      541 catttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
      601 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
      661 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
      721 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
      781 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
      841 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      901 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      961 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     1021 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     1081 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     1141 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     1201 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     1261 tgattctgtg gataaccgta ttaccgccat gcattagtta ttaatagtaa tcaattacgg
     1321 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     1381 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     1441 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     1501 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     1561 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     1621 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     1681 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     1741 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
     1801 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     1861 ctggtttagt gaagaagacg gctccggcgc cacaaacttc tctctgctaa agcaagcagg
     1921 tgatgttgaa gaaaaccccg ggcctggcag cggcgagggc aggggaagtc tacttacatg
     1981 cggggacgtg gaggaaaatc ccggcccagg atccgccgcc accatggtgt ctaagggcga
     2041 agagctgatc aaggaaaata tgcgtatgaa ggtggtcatg gaaggttcgg tcaacggcca
     2101 ccaattcaaa tgcacaggtg aaggagaagg cagaccgtac gagggagtgc aaaccatgag
     2161 gatcaaagtc atcgagggag gacccctgcc atttgccttt gacattcttg ccacgtcgtt
     2221 catgtatggc agccgtacct ttatcaagta cccggccgac atccctgatt tctttaaaca
     2281 gtcctttcct gagggtttta cttgggaaag agttacgaga tacgaagatg gtggagtcgt
     2341 caccgtcacg caggacacca gccttgagga tggcgagctc gtctacaacg tcaaggtcag
     2401 aggggtaaac tttccctcca atggtcccgt gatgcagaag aaaaccaagg gttgggagcc
     2461 taatacagag atgatgtatc cagcagatgg tggtctgaga ggatacactg acatcgcact
     2521 gaaagttgat ggtggtggcc atctgcactg caacttcgtg acaacttaca ggtcaaaaaa
     2581 gaccgtcggg aacatcaaga tgcccggtgt ccatgccgtt gatcaccgcc tggaaaggat
     2641 cgaggagagt gacaatgaaa cctacgtagt gcaaagagaa gtggcagttg ccaaatacag
     2701 caaccttggt ggtggcatgg acgagctgta caagtaacat ggtcgattca tgtcttctga
     2761 aacataaaat gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2821 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2881 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     2941 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     3001 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     3061 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     3121 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     3181 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     3241 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     3301 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcagta
     3361 cttaacggaa gatcacttcg tgatcggcac gtaagaggtt ccaactttca ccataatgaa
     3421 ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct
     3481 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa
     3541 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg
     3601 gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt
     3661 attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac
     3721 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgtttt
//
