LOCUS       sequence-452506-        3773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mClover3 and extra insert, for MuSIC barcode
            construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3773)
  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 3773)
  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-mClover3
FEATURES             Location/Qualifiers
     source          1..3773
                     /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             join(2024..2026,2027..2029,2030..2743)
                     /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(2069..2090)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2677..2698
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2778..2899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2906..3361)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3043..3064)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3255..3274
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3387..3489
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(3490..3773,1..376)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3556..3575)
                     /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 accatggtga gcaagggcga
     2041 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     2101 caagttcagc gtccgcggcg agggcgaggg cgatgccacc aacggcaagc tgaccctgaa
     2161 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg
     2221 ctacggcgtg gcctgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     2281 gtccgccatg cccgaaggct acgtccagga gcgcaccatc tctttcaagg acgacggtac
     2341 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     2401 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaactt
     2461 caacagccac tacgtctata tcacggccga caagcagaag aactgcatca aggctaactt
     2521 caagatccgc cacaacgttg aggacggcag cgtgcagctc gccgaccact accagcagaa
     2581 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gccatcagtc
     2641 caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     2701 cgccgccggg attacacatg gcatggacga gctgtacaag taaagatgtc gattcatgtc
     2761 ttctgaaaca taaaatgaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     2821 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     2881 aactcatcaa tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg
     2941 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     3001 tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt
     3061 ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat
     3121 ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca
     3181 ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac
     3241 gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta
     3301 gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg
     3361 tcagtactta acggaagatc acttcgtgat cggcacgtaa gaggttccaa ctttcaccat
     3421 aatgaaataa gatcactacc gggcgtattt tttgagttat cgagattttc aggagctaag
     3481 gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc ccaatggcat
     3541 cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa ccagaccgtt
     3601 cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa gttttatccg
     3661 gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattccg tatggcaatg
     3721 aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt ttt
//
