LOCUS       sequence-452500-        3773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mT-Sapphire 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-mT-Sapphire
FEATURES             Location/Qualifiers
     source          1..3773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     47..67
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             123..179
                     /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             189..242
                     /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      252..261
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(258..260,261..263,264..974)
                     /codon_start=1
                     /product="strongly enhanced green fluorescent protein 
                     (Kremers et al., 2007)"
                     /label=SGFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVMVFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSIQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(303..324)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(564..583)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     911..932
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1012..1133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1140..1595)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1277..1298)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1489..1508
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1621..1723
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1724..2383
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1790..1809)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     polyA_signal    2424..2545
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      2599..3187
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3088..3107
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3362..3665
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(3666..3773,1..96)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
       61 aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaagaa gacggctccg
      121 gcgccacaaa cttctctctg ctaaagcaag caggtgatgt tgaagaaaac cccgggcctg
      181 gcagcggcga gggcagggga agtctactta catgcgggga cgtggaggaa aatcccggcc
      241 caggatccgc cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
      301 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
      361 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
      421 ccgtgccctg gcccaccctc gtgaccacct tctcctacgg cgtgatggtg ttcgcccgct
      481 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
      541 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
      601 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
      661 gcaacatcct ggggcacaag ctggagtaca acttcaacag ccacaacgtc tatatcatgg
      721 ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg
      781 gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
      841 tgctgcccga caaccactac ctgagcatcc agtccaagct gagcaaagac cccaacgaga
      901 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
      961 acgagctgta caagtaagag ttgccattca tgtcttctga aacataaaat gaacttgttt
     1021 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     1081 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta
     1141 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     1201 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     1261 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     1321 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     1381 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     1441 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     1501 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     1561 acccgccgcg cttaatgcgc cgctacaggg cgcgtcagta cttaacggaa gatcacttcg
     1621 tgatcggcac gtaagaggtt ccaactttca ccataatgaa ataagatcac taccgggcgt
     1681 attttttgag ttatcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac
     1741 tggatatacc accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca
     1801 gtcagttgct caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa
     1861 gaccgtaaag aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct
     1921 gatgaatgct catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga
     1981 tagtgttcac ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg
     2041 gagtgaatac cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg
     2101 ttacggtgaa aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtatc
     2161 agccaatccc tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt
     2221 cttcgccccc gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc
     2281 gctggcgatt caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa
     2341 tgaattacaa cagtactgcg atgagtggca gggcggggcg taatcaaaca tgagaattgg
     2401 tcgcgcgagc aattcagaca tgataagata cattgatgag tttggacaaa ccacaactag
     2461 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     2521 cattataagc tgcaataaac aagttggcat ttcgttccac tgagcgtcag accccgtaga
     2581 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     2641 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     2701 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     2761 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     2821 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     2881 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     2941 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     3001 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     3061 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     3121 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     3181 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     3241 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca
     3301 ttagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     3361 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     3421 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     3481 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     3541 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     3601 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     3661 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     3721 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caa
//
