LOCUS       sequence-474985-        5349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the I3-01 nanocage subunit C-terminally tagged with EGFP 
            in mammalian cells. Assembles into nanocages tagged with 60 EGFPs..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5349)
  AUTHORS   Viola G, Ibrahim YW, Jacobs KA, Lemiere J, Kutys ML, Wittmann T
  TITLE     Quantitative comparison of fluorescent proteins using protein 
            nanocages in live cells.
  JOURNAL   J Cell Sci. 2025 May 15;138(10):jcs263858. doi: 10.1242/jcs.263858. 
            Epub 2025 May 21.
  PUBMED    40326462
REFERENCE   2  (bases 1 to 5349)
  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"; journalName: "J Cell 
            Sci."; date: "2025-05-15"; volume: "138(10):jcs263858. doi"; pages: 
            " 10.1242/jcs.263858. Epub 2025 May 21"
COMMENT     Sequence Label: pI3_EGFP
FEATURES             Location/Qualifiers
     source          1..5349
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        764..1067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1068..1271
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1222..1242
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(2009..2011,2012..2725)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2009..2011,2012..2722)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2051..2072)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2312..2331)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2659..2680
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2840..2961
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2877..2896)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2931..2950
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2968..3423)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3105..3126)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3317..3336
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3450..3554
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3556..3913
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3556..3747
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3579..3599)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3764..3899
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3948..4742
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3948..4742
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4002..4021)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4612..4631
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4930..4949)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4974..5021
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
      721 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      781 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      841 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      901 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      961 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     1021 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
     1081 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
     1141 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
     1201 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
     1261 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact cagatctcga
     1321 gctcaagctt cgaattctgc agtcgacggt accgcgggcc cggatgaaaa tggaggaact
     1381 ctttaagaag cataaaatcg tagctgtgct cagagcaaat tcagtagagg aggcgaagaa
     1441 aaaggccctg gccgttttcc tgggcggagt ccacctcatc gaaattactt ttactgtccc
     1501 cgacgcagat acagtcataa aagagttgag ttttctcaaa gagatggggg cgatcatcgg
     1561 tgccgggacg gttacgtcag tcgagcagtg ccggaaagca gtggagtcag gagcggaatt
     1621 tatcgtatct ccacatctgg atgaggaaat atctcaattc tgtaaagaaa aaggtgtctt
     1681 ttacatgccc ggcgttatga cgccaacgga gctggtgaag gccatgaagc tgggacacac
     1741 gatactggct ctctttccag gcgaagtggt cggtcctcaa ttcgtcaagg caatgaaagg
     1801 gcctttcccc aacgttaaat tcgtcccgac tgggggagta aacttggaca atgtttgcga
     1861 atggtttaag gcaggggtac tcgctgttgg agttggttcc gcattggtaa aaggtacgcc
     1921 tgtcgaagtg gcagaaaagg caaaggcttt cgttgagaag atccgcggat gtactgaagg
     1981 tagcggttca gggagtggta gcggcagtgt gagcaagggc gaggagctgt tcaccggggt
     2041 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     2101 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     2161 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     2221 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     2281 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     2341 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     2401 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     2461 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     2521 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     2581 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     2641 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     2701 cggcatggac gagctgtaca agtagcgact ctagatcata atcagccata ccacatttgt
     2761 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     2821 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     2881 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     2941 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     3001 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     3061 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     3121 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     3181 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     3241 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     3301 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     3361 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     3421 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     3481 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3541 gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     3601 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3661 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3721 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     3781 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     3841 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     3901 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3961 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     4021 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     4081 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     4141 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     4201 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     4261 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     4321 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     4381 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4441 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     4501 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4561 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4621 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4681 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4741 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4801 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4861 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     4921 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     4981 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     5041 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     5101 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     5161 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     5221 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     5281 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     5341 aggatcttc
//
