LOCUS       sequence-452490-        3764 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of mOrange2 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-mOrange2
FEATURES             Location/Qualifiers
     source          1..3764
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     44..64
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             120..176
                     /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             186..239
                     /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      249..258
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             255..965
                     /codon_start=1
                     /product="photostable monomeric orange derivative of DsRed 
                     fluorescent protein (Shaner et al., 2008)"
                     /label=mOrange2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTAKLKVTKGGPLPFAWDILSPHFTYGSKAYVKHPADIPDYFKLSFPEGFKWERVMNY
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GKIKMRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     polyA_signal    1000..1121
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1128..1583)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1265..1286)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1477..1496
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1609..1711
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1712..2371
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1778..1797)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     polyA_signal    2412..2533
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      2587..3175
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3076..3095
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3350..3653
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(3654..3764,1..93)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
       61 cgtgtacggt gggaggtcta tataagcaga gctggtttag tgaagaagac ggctccggcg
      121 ccacaaactt ctctctgcta aagcaagcag gtgatgttga agaaaacccc gggcctggca
      181 gcggcgaggg caggggaagt ctacttacat gcggggacgt ggaggaaaat cccggcccag
      241 gatccgccgc caccatggtg agcaagggcg aggagaataa catggccatc atcaaggagt
      301 tcatgcgctt caaggtgcgc atggagggct ccgtgaacgg ccacgagttc gagatcgagg
      361 gcgagggcga gggccgcccc tacgagggct ttcagaccgc taagctgaag gtgaccaagg
      421 gtggccccct gcccttcgcc tgggacatcc tgtcccctca tttcacctac ggctccaagg
      481 cctacgtgaa gcaccccgcc gacatccccg actacttcaa gctgtccttc cccgagggct
      541 tcaagtggga gcgcgtgatg aactacgagg acggcggcgt ggtgaccgtg acccaggact
      601 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
      661 ccgacggccc cgtgatgcag aagaaaacca tgggctggga ggcctcctcc gagcggatgt
      721 accccgagga cggtgccctg aagggcaaga tcaagatgag gctgaagctg aaggacggcg
      781 gccactacac ctccgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
      841 gcgcctacat cgtcgacatc aagttggaca tcacctccca caacgaggac tacaccatcg
      901 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
      961 agtaagagtt gccattcatg tcttctgaaa cataaaatga acttgtttat tgcagcttat
     1021 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     1081 cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt
     1141 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     1201 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     1261 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     1321 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     1381 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     1441 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     1501 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     1561 taatgcgccg ctacagggcg cgtcagtact taacggaaga tcacttcgtg atcggcacgt
     1621 aagaggttcc aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt
     1681 atcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac
     1741 cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca
     1801 atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa
     1861 aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca
     1921 tccggaattc cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc
     1981 ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca
     2041 cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa
     2101 cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtatcag ccaatccctg
     2161 ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt
     2221 tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca
     2281 ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca
     2341 gtactgcgat gagtggcagg gcggggcgta atcaaacatg agaattggtc gcgcgagcaa
     2401 ttcagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     2461 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     2521 caataaacaa gttggcattt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2581 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2641 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2701 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     2761 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2821 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2881 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     2941 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3001 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3061 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3121 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3181 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3241 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gccatgcatt agttattaat
     3301 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     3361 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     3421 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     3481 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     3541 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     3601 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
     3661 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
     3721 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaat
//
