LOCUS       sequence-452497-        3773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EBFP2 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-EBFP2
FEATURES             Location/Qualifiers
     source          1..3773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(55..768,769..771,772..774))
                     /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     708..729
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      771..780
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(790..843)
                     /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"
     CDS             complement(853..909)
                     /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"
     promoter        complement(936..1139)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(965..985)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1140..1443
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(1618..2206)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1698..1717)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    2260..2381
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(2422..3081)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     2996..3015
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3082..3184)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3210..3665
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3297..3316)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3507..3528
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    join(3672..3773,1..20)
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
ORIGIN
        1 taagctgcaa taaacaagtt cattttatgt ttcagaagac atgaatggca actcttactt
       61 gtacagctcg tccatgccga gagtgatccc ggcggcggtg cggaactcca gcaggaccat
      121 gtgatcgcgc ttctcgttgg ggtctttgct cagcacggac tgggtgctca ggtagtggct
      181 gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag
      241 ctgcacgctg ccgtcctcca cgttgtggcg gatcttgaag ttcaccttga tgccgttctt
      301 ctgcttgacg gccatgatat agatgttgtg gctgttgaag ttgtactcca gcttgtgccc
      361 caggatgttg ccgtcctcct tgaagtcgac gcccttcagc tcgatgcggt tcactagggt
      421 gtcgccctcg aacttcacct cggcgcgggt cttgtaggtg ccgtcgtcct tgaagaagat
      481 ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt gctgcttcat
      541 gtggtcgggg tagcgggcga agcactgcac gccgtggctc agggtggtca cgagggtggg
      601 ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct tgccgttggt
      661 ggcatcgccc tcgccctcgc ccctcacgct gaacttgtgg ccgtttacgt cgccgtccag
      721 ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca ccatggtggc
      781 ggcggatcct gggccgggat tttcctccac gtccccgcat gtaagtagac ttcccctgcc
      841 ctcgccgctg ccaggcccgg ggttttcttc aacatcacct gcttgcttta gcagagagaa
      901 gtttgtggcg ccggagccgt cttcttcact aaaccagctc tgcttatata gacctcccac
      961 cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag
     1021 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
     1081 aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc
     1141 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
     1201 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
     1261 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
     1321 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
     1381 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
     1441 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
     1501 ctaatgcatg gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     1561 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1621 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     1681 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     1741 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     1801 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     1861 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     1921 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     1981 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2041 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2101 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2161 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2221 cttttctacg gggtctgacg ctcagtggaa cgaaatgcca acttgtttat tgcagcttat
     2281 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     2341 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg aattgctcgc
     2401 gcgaccaatt ctcatgtttg attacgcccc gccctgccac tcatcgcagt actgttgtaa
     2461 ttcattaagc attctgccga catggaagcc atcacaaacg gcatgatgaa cctgaatcgc
     2521 cagcggcatc agcaccttgt cgccttgcgt ataatatttg cccatggtga aaacgggggc
     2581 gaagaagttg tccatattgg ccacgtttaa atcaaaactg gtgaaactca cccagggatt
     2641 ggctgatacg aaaaacatat tctcaataaa ccctttaggg aaataggcca ggttttcacc
     2701 gtaacacgcc acatcttgcg aatatatgtg tagaaactgc cggaaatcgt cgtggtattc
     2761 actccagagc gatgaaaacg tttcagtttg ctcatggaaa acggtgtaac aagggtgaac
     2821 actatcccat atcaccagct caccgtcttt cattgccata cggaattccg gatgagcatt
     2881 catcaggcgg gcaagaatgt gaataaaggc cggataaaac ttgtgcttat ttttctttac
     2941 ggtctttaaa aaggccgtaa tatccagctg aacggtctgg ttataggtac attgagcaac
     3001 tgactgaaat gcctcaaaat gttctttacg atgccattgg gatatatcaa cggtggtata
     3061 tccagtgatt tttttctcca ttttagcttc cttagctcct gaaaatctcg ataactcaaa
     3121 aaatacgccc ggtagtgatc ttatttcatt atggtgaaag ttggaacctc ttacgtgccg
     3181 atcacgaagt gatcttccgt taagtactga cgcgccctgt agcggcgcat taagcgcggc
     3241 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3301 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3361 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3421 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3481 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3541 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3601 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac
     3661 aatttacgcc ttaagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     3721 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca tta
//
