LOCUS       sequence-452482-        3773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of EGFP 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-EGFP
FEATURES             Location/Qualifiers
     source          1..3773
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     15..34
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(101..203)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      229..684
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(316..335)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     526..547
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    691..812
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(847..1560,1561..1563,1564..1566))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(892..913)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1241..1260
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1500..1521
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1563..1572
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1582..1635)
                     /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"
     CDS             complement(1645..1701)
                     /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"
     promoter        complement(1728..1931)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1757..1777)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1932..2235
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(2410..2998)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2490..2509)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3052..3173
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(join(3214..3773,1..100))
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 tataggtaca ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg
       61 atatatcaac ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg
      121 aaaatctcga taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt
      181 tggaacctct tacgtgccga tcacgaagtg atcttccgtt aagtactgac gcgccctgta
      241 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
      301 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
      361 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
      421 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
      481 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
      541 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
      601 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
      661 acaaaatatt aacgcttaca atttacgcct taagatacat tgatgagttt ggacaaacca
      721 caactagaat gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat
      781 ttgtaaccat tataagctgc aataaacaag ttcattttat gtttcagaag acatgaatca
      841 tacagtttac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc
      901 cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagcttgg actgggtgct
      961 caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt tctgctggta
     1021 gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga agttcacctt
     1081 gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt agttgtactc
     1141 cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg
     1201 gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc
     1261 cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact tgaagaagtc
     1321 gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg tcagggtggt
     1381 cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact tcagggtcag
     1441 cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt ggccgtttac
     1501 gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct cgcccttgct
     1561 caccatggtg gcggcggatc ctgggccggg attttcctcc acgtccccgc atgtaagtag
     1621 acttcccctg ccctcgccgc tgccaggccc ggggttttct tcaacatcac ctgcttgctt
     1681 tagcagagag aagtttgtgg cgccggagcc gtcttcttca ctaaaccagc tctgcttata
     1741 tagacctccc accgtacacg cctaccgccc atttgcgtca atggggcgga gttgttacga
     1801 cattttggaa agtcccgttg attttggtgc caaaacaaac tcccattgac gtcaatgggg
     1861 tggagacttg gaaatccccg tgagtcaaac cgctatccac gcccattgat gtactgccaa
     1921 aaccgcatca ccatggtaat agcgatgact aatacgtaga tgtactgcca agtaggaaag
     1981 tcccataagg tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca
     2041 atagggggcg tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt
     2101 aaatactcca cccattgacg tcaatggaaa gtccctattg gcgttactat gggaacatac
     2161 gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc
     2221 gtaagttatg taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat
     2281 tactattaat aactaatgca tggcggtaat acggttatcc acagaatcag gggataacgc
     2341 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     2401 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2461 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2521 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2581 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2641 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2701 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     2761 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     2821 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     2881 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2941 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3001 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaatgc caacttgttt
     3061 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3121 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     3181 tgaattgctc gcgcgaccaa ttctcatgtt tgattacgcc ccgccctgcc actcatcgca
     3241 gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaaa cggcatgatg
     3301 aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatggt
     3361 gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt aaatcaaaac tggtgaaact
     3421 cacccaggga ttggctgata cgaaaaacat attctcaata aaccctttag ggaaataggc
     3481 caggttttca ccgtaacacg ccacatcttg cgaatatatg tgtagaaact gccggaaatc
     3541 gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt tgctcatgga aaacggtgta
     3601 acaagggtga acactatccc atatcaccag ctcaccgtct ttcattgcca tacggaattc
     3661 cggatgagca ttcatcaggc gggcaagaat gtgaataaag gccggataaa acttgtgctt
     3721 atttttcttt acggtcttta aaaaggccgt aatatccagc tgaacggtct ggt
//
