LOCUS       sequence-457663-        3764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of LSSmOrange and extra insert, for MuSIC 
            barcode construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3764)
  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 3764)
  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-LSSmOrange
FEATURES             Location/Qualifiers
     source          1..3764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    415..536
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      590..1178
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1079..1098
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1353..1656
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1657..1860
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1811..1831
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             1887..1943
                     /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             1953..2006
                     /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      2016..2025
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2022..2732
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     polyA_signal    2767..2888
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(2895..3350)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3032..3053)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3244..3263
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3376..3478
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             join(3479..3764,1..374)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3545..3564)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
ORIGIN
        1 atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt
       61 ttctacacat atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta
      121 aagggtttat tgagaatatg tttttcgtat cagccaatcc ctgggtgagt ttcaccagtt
      181 ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc atgggcaaat
      241 attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt
      301 gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc
      361 agggcggggc gtaatcaaac atgagaattg gtcgcgcgag caattcagac atgataagat
      421 acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg
      481 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttggca
      541 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
      601 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
      661 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
      721 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
      781 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
      841 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
      901 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
      961 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     1021 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     1081 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     1141 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     1201 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     1261 attctgtgga taaccgtatt accgccatgc attagttatt aatagtaatc aattacgggg
     1321 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     1381 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     1441 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1501 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     1561 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     1621 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     1681 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     1741 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     1801 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     1861 ggtttagtga agaagacggc tccggcgcca caaacttctc tctgctaaag caagcaggtg
     1921 atgttgaaga aaaccccggg cctggcagcg gcgagggcag gggaagtcta cttacatgcg
     1981 gggacgtgga ggaaaatccc ggcccaggat ccgccgccac catggtgagc aagggcgagg
     2041 agaataacat ggccatcatc aaggagttca tgcgcttcaa ggtgcgcatg gagggctccg
     2101 tgaacggcca cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggctttc
     2161 agaccgttaa gctgaaggtt accaagggtg gaccactgcc tttcgcctgg gacatattgt
     2221 cacctcagtt cacctacggc tccaaggcct acgtgaagca ccccgccgac atccccgact
     2281 acctcaagct gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggatg
     2341 gcggcgtggt gaccgtgact caggactcct ccctgcagga cggcgagttc atctacaagg
     2401 tgaagctgcg cggcaccaac ttcccctccg acggacccgt aatgcagaag aaaaccatgg
     2461 gcatggaagc ctcatctgaa cggatgtacc ccgaggacgg cgcactgaag ggcgaggaca
     2521 agctcaggct gaagctgaag gacggcggcc actacacctc cgaggtcaag accacctaca
     2581 aggccaagaa gcccgtgcag ttgccaggcg cctacatcgt cgacatcaag ttggacatca
     2641 cctcacacaa cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact
     2701 ccaccggcgg catggacgag ctgtacaagt aagagttgcc attcatgtct tctgaaacat
     2761 aaaatgaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     2821 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     2881 gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg
     2941 ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa
     3001 agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa
     3061 gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg
     3121 tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa
     3181 ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa
     3241 ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct
     3301 gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cagtacttaa
     3361 cggaagatca cttcgtgatc ggcacgtaag aggttccaac tttcaccata atgaaataag
     3421 atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg aagctaaaat
     3481 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
     3541 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
     3601 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
     3661 cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga aagacggtga
     3721 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttcc
//
