LOCUS       sequence-452498-        3773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mCerulean3 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-mCerulean3
FEATURES             Location/Qualifiers
     source          1..3773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    420..541
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      595..1183
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1084..1103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1358..1661
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1662..1865
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1816..1836
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1892..1948
                     /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             1958..2011
                     /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      2021..2030
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2027..2029,2030..2032,2033..2746)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAIHGNVYITADKQKNGIK
                     ANFGLNCNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2072..2093)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2333..2352)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2680..2701
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2781..2902
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2909..3364)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3046..3067)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3258..3277
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3390..3492
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(3493..3773,1..379)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3559..3578)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
       61 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
      121 ccctaaaggg tttattgaga atatgttttt cgtatcagcc aatccctggg tgagtttcac
      181 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
      241 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
      301 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
      361 gtggcagggc ggggcgtaat caaacatgag aattggtcgc gcgagcaatt cagacatgat
      421 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
      481 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
      541 tggcatttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
      601 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
      661 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
      721 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
      781 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
      841 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
      901 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
      961 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     1021 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     1081 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     1141 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     1201 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc
     1261 ccctgattct gtggataacc gtattaccgc catgcattag ttattaatag taatcaatta
     1321 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1381 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1441 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1501 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1561 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1621 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1681 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1741 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1801 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1861 gagctggttt agtgaagaag acggctccgg cgccacaaac ttctctctgc taaagcaagc
     1921 aggtgatgtt gaagaaaacc ccgggcctgg cagcggcgag ggcaggggaa gtctacttac
     1981 atgcggggac gtggaggaaa atcccggccc aggatccgcc gccaccatgg tgagcaaggg
     2041 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     2101 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     2161 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     2221 gagctggggc gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt
     2281 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     2341 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     2401 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     2461 cgccatccac ggcaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa
     2521 cttcggcctc aactgcaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     2581 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     2641 gtccaagctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     2701 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagta gtaaattcat
     2761 gtcttctgaa acataaaatg aacttgttta ttgcagctta taatggttac aaataaagca
     2821 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     2881 ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc
     2941 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     3001 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     3061 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     3121 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     3181 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     3241 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     3301 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     3361 gcgtcagtac ttaacggaag atcacttcgt gatcggcacg taagaggttc caactttcac
     3421 cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt ttcaggagct
     3481 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
     3541 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
     3601 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
     3661 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt ccgtatggca
     3721 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgt
//
