LOCUS       sequence-452499-        3773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mAmetrine 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-mAmetrine
FEATURES             Location/Qualifiers
     source          1..3773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    416..537
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      591..1179
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1080..1099
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1354..1657
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1658..1861
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1812..1832
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1888..1944
                     /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             1954..2007
                     /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      2017..2026
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2023..2025,2026..2028,2029..2742)
                     /codon_start=1
                     /product="yellow fluorescent protein with a large Stokes 
                     shift, excitable with violet light (Ai et al., 2008)"
                     /label=mAmetrine
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTSGKLPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GSYRTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNMNVWDAYITADKQKNGIK
                     ANFKIEHNVEDGGVQLADAYQQNTPIGDGSVLLPDNHYLSFQSKLFKDPNEQRDHMVLL
                     EFVTAAGITPGMDELYK"
     primer_bind     complement(2068..2089)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    2777..2898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2905..3360)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3042..3063)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3254..3273
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3386..3488
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(3489..3773,1..375)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3555..3574)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag
       61 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct
      121 aaagggttta ttgagaatat gtttttcgta tcagccaatc cctgggtgag tttcaccagt
      181 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa
      241 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt
      301 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg
      361 cagggcgggg cgtaatcaaa catgagaatt ggtcgcgcga gcaattcaga catgataaga
      421 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt
      481 gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa acaagttggc
      541 atttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
      601 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
      661 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
      721 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
      781 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
      841 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
      901 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
      961 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     1021 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     1081 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     1141 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     1201 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     1261 gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat caattacggg
     1321 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     1381 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     1441 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     1501 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     1561 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     1621 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     1681 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     1741 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     1801 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     1861 tggtttagtg aagaagacgg ctccggcgcc acaaacttct ctctgctaaa gcaagcaggt
     1921 gatgttgaag aaaaccccgg gcctggcagc ggcgagggca ggggaagtct acttacatgc
     1981 ggggacgtgg aggaaaatcc cggcccagga tccgccgcca ccatggtgag caagggcgag
     2041 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     2101 aagttcagcg tgcgcggcga gggcgagggc gatgccacca acggcaagct gaccctgaag
     2161 ttcatctgca cctccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgtct
     2221 tacggcgtgc agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag
     2281 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct ccttcaagga cgacggcagc
     2341 tacaggaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     2401 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaacatg
     2461 aacgtgtggg acgcgtatat cacggccgac aagcagaaga acggcatcaa agcgaacttc
     2521 aagatcgagc acaacgtcga ggacggcggc gtgcagctcg ccgacgcgta ccagcagaac
     2581 acccccatcg gcgacggctc cgtgctgctg cctgacaacc actacctgag cttccagagc
     2641 aagctgttca aagaccccaa cgagcagcgc gatcacatgg tcctgctgga gttcgttacc
     2701 gccgccggga tcactcccgg catggacgag ctgtacaagt aagagttgcc attcatgtct
     2761 tctgaaacat aaaatgaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     2821 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     2881 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     2941 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3001 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3061 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3121 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3181 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3241 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3301 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3361 cagtacttaa cggaagatca cttcgtgatc ggcacgtaag aggttccaac tttcaccata
     3421 atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg
     3481 aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc
     3541 gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc
     3601 agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg
     3661 cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga
     3721 aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttc
//
