LOCUS       sequence-452485-        4001 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of miRFP670 and extra insert, for MuSIC barcode
            construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4001)
  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 4001)
  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-miRFP670
FEATURES             Location/Qualifiers
     source          1..4001
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     19..39
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             95..151
                     /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             161..214
                     /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      224..233
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             230..1177
                     /codon_start=1
                     /product="bright monomeric near-infrared fluorescent 
                     protein engineered from a bacterial phytochrome, with an 
                     emission maximum of 670 nm (Shcherbakova et al., 2016)"
                     /label=miRFP670
                     /label=ORF frame 1
                     /translation="MVAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDH
                     RVIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTE
                     YCGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTG
                     YDRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVT
                     YQPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLV
                     VCHHYLPRFIRFELRAICKRLAERIATRITALES"
     polyA_signal    1212..1333
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      complement(1340..1795)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1477..1498)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1689..1708
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1821..1923
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1924..2583
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1990..2009)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     polyA_signal    2624..2745
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      2799..3387
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3288..3307
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        3562..3865
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(3866..4001,1..68)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
ORIGIN
        1 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
       61 gcagagctgg tttagtgaag aagacggctc cggcgccaca aacttctctc tgctaaagca
      121 agcaggtgat gttgaagaaa accccgggcc tggcagcggc gagggcaggg gaagtctact
      181 tacatgcggg gacgtggagg aaaatcccgg cccaggatcc gccgccacca tggtagcagg
      241 tcatgcctct ggcagccccg cattcgggac cgcctctcat tcgaattgcg aacatgaaga
      301 gatccacctc gccggctcga tccagccgca tggcgcgctt ctggtcgtca gcgaacatga
      361 tcatcgcgtc atccaggcca gcgccaacgc cgcggaattt ctgaatctcg gaagcgtact
      421 cggcgttccg ctcgccgaga tcgacggcga tctgttgatc aagatcctgc cgcatctcga
      481 tcccaccgcc gaaggcatgc cggtcgcggt gcgctgccgg atcggcaatc cctctacgga
      541 gtactgcggt ctgatgcatc ggcctccgga aggcgggctg atcatcgaac tcgaacgtgc
      601 cggcccgtcg atcgatctgt caggcacgct ggcgccggcg ctggagcgga tccgcacggc
      661 gggttcactg cgcgcgctgt gcgatgacac cgtgctgctg tttcagcagt gcaccggcta
      721 cgaccgggtg atggtgtatc gtttcgatga gcaaggccac ggcctggtat tctccgagtg
      781 ccatgtgcct gggctcgaat cctatttcgg caaccgctat ccgtcgtcga ctgtcccgca
      841 gatggcgcgg cagctgtacg tgcggcagcg cgtccgcgtg ctggtcgacg tcacctatca
      901 gccggtgccg ctggagccgc ggctgtcgcc gctgaccggg cgcgatctcg acatgtcggg
      961 ctgcttcctg cgctcgatgt cgccgtgcca tctgcagttc ctgaaggaca tgggcgtgcg
     1021 cgccaccctg gcggtgtcgc tggtggtcgg cggcaagctg tggggcctgg ttgtctgtca
     1081 ccattatctg ccgcgcttca tccgtttcga gctgcgggcg atctgcaaac ggctcgccga
     1141 aaggatcgcg acgcggatca ccgcgcttga gagctaagag ttgccattca tgtcttctga
     1201 aacataaaat gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     1261 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     1321 tcaatgtatc ttaaggcgta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     1381 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     1441 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     1501 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     1561 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     1621 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     1681 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     1741 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcagta
     1801 cttaacggaa gatcacttcg tgatcggcac gtaagaggtt ccaactttca ccataatgaa
     1861 ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct
     1921 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa
     1981 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg
     2041 gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt
     2101 attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac
     2161 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact
     2221 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata
     2281 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt
     2341 gagaatatgt ttttcgtatc agccaatccc tgggtgagtt tcaccagttt tgatttaaac
     2401 gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa
     2461 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc
     2521 catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg
     2581 taatcaaaca tgagaattgg tcgcgcgagc aattcagaca tgataagata cattgatgag
     2641 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     2701 gctattgctt tatttgtaac cattataagc tgcaataaac aagttggcat ttcgttccac
     2761 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2821 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2881 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2941 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3001 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3061 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     3121 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     3181 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     3241 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     3301 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     3361 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     3421 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     3481 aaccgtatta ccgccatgca ttagttatta atagtaatca attacggggt cattagttca
     3541 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     3601 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     3661 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     3721 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     3781 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     3841 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     3901 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     3961 gttttggcac caaaatcaac gggactttcc aaaatgtcgt a
//
