LOCUS       sequence-428729-        5114 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Parental minicircle containing EGFP flanked by a splice acceptor and
            splice donor. Used with other intron tagging plasmids for placing 
            the EGFP tag as a synthetic exon into frame 0 introns of target 
            genes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5114)
  AUTHORS   Reicher A, Reinis J, Ciobanu M, Ruzicka P, Malik M, Siklos M, 
            Kartysh V, Tomek T, Koren A, Rendeiro AF, Kubicek S
  TITLE     Pooled multicolour tagging for visualizing subcellular protein 
            dynamics.
  JOURNAL   Nat Cell Biol. 2024 May;26(5):745-756. doi: 
            10.1038/s41556-024-01407-w. Epub 2024 Apr 19.
  PUBMED    38641660
REFERENCE   2  (bases 1 to 5114)
  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: 
            "10.1038/s41556-024-01407-w"; journalName: "Nat Cell Biol"; date: 
            "2024-05"; volume: "26"; issue: "5"; pages: "745-756"
COMMENT     Sequence Label: Intron-Tagging-EGFP-Frame0-Parental-Minicircle
FEATURES             Location/Qualifiers
     source          1..5114
                     /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"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..915
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(841..860)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1001..1023
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2254..2271)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    2292..2325
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             join(2488..2490,2491..3201)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2488..2490,2491..3201)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2530..2551)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2791..2810)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3138..3159
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(3448..4242)
                     /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             complement(3448..4242)
                     /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(3559..3578)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4169..4188
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4606..4623)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4776..4862
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4954..4981
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct 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 taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag
      841 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      901 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      961 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     1021 aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg
     1081 aagcggcatg cataatgtgc ctgtcaaatg gacgaagcag ggattctgca aaccctatgc
     1141 tactccgtca agccgtcaat tgtctgattc gttaccaatt atgacaactt gacggctaca
     1201 tcattcactt tttcttcaca accggcacgg aactcgctcg ggctggcccc ggtgcatttt
     1261 ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg
     1321 ataggcatcc gggtggtgct caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc
     1381 cagcttaaga cgctaatccc taactgctgg cggaaaagat gtgacagacg cgacggcgac
     1441 aagcaaacat gctgtgcgac gctggcgatt cttctctcat ccgccaaaac agccaagctg
     1501 gagaccgttt gacattaccc tgttatccct agatacatta ccctgttatc ccagatgaca
     1561 taccctgtta tccctagatg acattaccct gttatcccag atgacattac cctgttatcc
     1621 ctagatacat taccctgtta tcccagatga cataccctgt tatccctaga tgacattacc
     1681 ctgttatccc agatgacatt accctgttat ccctagatac attaccctgt tatcccagat
     1741 gacataccct gttatcccta gatgacatta ccctgttatc ccagatgaca ttaccctgtt
     1801 atccctagat acattaccct gttatcccag atgacatacc ctgttatccc tagatgacat
     1861 taccctgtta tcccagatga cattaccctg ttatccctag atacattacc ctgttatccc
     1921 agatgacata ccctgttatc cctagatgac attaccctgt tatcccagat gacattaccc
     1981 tgttatccct agatacatta ccctgttatc ccagatgaca taccctgtta tccctagatg
     2041 acattaccct gttatcccag atgacattac cctgttatcc ctagatacat taccctgtta
     2101 tcccagatga cataccctgt tatccctaga tgacattacc ctgttatccc agatgacatt
     2161 accctgttat ccctagatac attaccctgt tatcccagat gacataccct gttatcccta
     2221 gatgacatta ccctgttatc ccagataaac tcaatgatga tgatgatgat ggtcgagact
     2281 cagcggccgc ggtgccaggg cgtgcccttg ggctccccgg gcgcgactag tgaattcaga
     2341 tctgatcctg cgttatcccc tggagatcga gtgccgcatc accggctatt ggtcttactg
     2401 acatccactt tgcctttctc tccacagggt ggcggtggct cgggcggtgg tgggtccggt
     2461 ggcggcggat ctggcggtgg tggatccgtg agcaagggcg aggagctgtt caccggggtg
     2521 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     2581 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     2641 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     2701 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     2761 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     2821 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     2881 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     2941 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     3001 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3061 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     3121 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3181 ggcatggacg agctgtacaa gggcggcggt ggatccggtg gcggcggatc tggcggtgga
     3241 ggttcgggtg gcggtgggtc gggcaaggta agtatcaatt agaagacgaa ttccatctct
     3301 agagtcgacc catgggggcc cgccccaact ggggtaacct ttgagttctc tcagttgggg
     3361 gtaatcagca tcatgatgtg gtaccacatc atgatgctga ttataagaat gcggccgcca
     3421 cactctagtg gatctcgagt taataattca gaagaactcg tcaagaaggc gatagaaggc
     3481 gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt cagcccattc
     3541 gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat agcggtccgc
     3601 cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca ccatgatatt
     3661 cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca tgctcgcctt
     3721 gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg
     3781 atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg
     3841 gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat cagccatgat
     3901 ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg gcacttcgcc
     3961 caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac
     4021 gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct tgcagttcat tcagggcacc
     4081 ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc
     4141 ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc tctccaccca
     4201 agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc
     4261 tgtctcttga tcagagcttg atcccctgcg ccatcagatc cttggcggca agaaagccat
     4321 ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg gcaattccgg
     4381 ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc cactgcaagc
     4441 tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt agctgacatt
     4501 catccggggt cagcaccgtt tctgcggact ggctttctac gcgctcgagg gggggccaaa
     4561 cggtctccag cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa
     4621 atcagaacgc agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt
     4681 cccacctgac cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg
     4741 gtctccccat gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga
     4801 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
     4861 atccgccggg agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac
     4921 gcccgccata aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt
     4981 ttgcgtttct acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc
     5041 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5101 atcaaaggat cttc
//
