LOCUS       sequence-428733-        5096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Parental minicircle containing mScarlet-I flanked by a splice 
            acceptor and splice donor. Used with other intron tagging plasmids 
            for placing the mScarlet tag as a synthetic exon into frame 1 
            introns of target genes..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5096)
  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 5096)
  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-mScarlet-Frame1-Parental-Minici...
FEATURES             Location/Qualifiers
     source          1..5096
                     /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             2490..3182
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(2620..2638)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2888..2907
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             complement(3430..4224)
                     /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(3430..4224)
                     /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(3541..3560)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4151..4170
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4588..4605)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4758..4844
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4936..4963
                     /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 tccacagggg gtggcggtgg ctcgggcggt ggtgggtccg
     2461 gtggcggcgg atctggcggt ggtggatccg tgagcaaggg cgaggcagtg atcaaggagt
     2521 tcatgcggtt caaggtgcac atggagggct ccatgaacgg ccacgagttc gagatcgagg
     2581 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     2641 gtggccccct gcccttctcc tgggacatcc tgtcccctca gttcatgtac ggctccaggg
     2701 ccttcatcaa gcaccccgcc gacatccccg actactataa gcagtccttc cccgagggct
     2761 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgc cgtgaccgtg acccaggaca
     2821 cctccctgga ggacggcacc ctgatctaca aggtgaagct ccgcggcacc aacttccctc
     2881 ctgacggccc cgtaatgcag aagaagacaa tgggctggga agcgtccacc gagcggttgt
     2941 accccgagga cggcgtgctg aagggcgaca ttaagatggc cctgcgcctg aaggacggcg
     3001 gccgctacct ggcggacttc aagaccacct acaaggccaa gaagcccgtg cagatgcccg
     3061 gcgcctacaa cgtcgaccgc aagttggaca tcacctccca caacgaggac tacaccgtgg
     3121 tggaacagta cgaacgctcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     3181 agggcggcgg tggatccggt ggcggcggat ctggcggtgg aggttcgggt ggcggtgggt
     3241 cgggcggagg taagtatcaa ttagaagacg aattccatct ctagagtcga cccatggggg
     3301 cccgccccaa ctggggtaac ctttgagttc tctcagttgg gggtaatcag catcatgatg
     3361 tggtaccaca tcatgatgct gattataaga atgcggccgc cacactctag tggatctcga
     3421 gttaataatt cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg
     3481 agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc
     3541 aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca
     3601 gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc
     3661 atgggtcacg acgagatcct cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc
     3721 ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc
     3781 catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc
     3841 cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg
     3901 agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct
     3961 tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca
     4021 cgatagccgc gctgcctcgt cttgcagttc attcagggca ccggacaggt cggtcttgac
     4081 aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat
     4141 tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc
     4201 gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagagct
     4261 tgatcccctg cgccatcaga tccttggcgg caagaaagcc atccagttta ctttgcaggg
     4321 cttcccaacc ttaccagagg gcgccccagc tggcaattcc ggttcgcttg ctgtccataa
     4381 aaccgcccag tctagctatc gccatgtaag cccactgcaa gctacctgct ttctctttgc
     4441 gcttgcgttt tcccttgtcc agatagccca gtagctgaca ttcatccggg gtcagcaccg
     4501 tttctgcgga ctggctttct acgcgctcga gggggggcca aacggtctcc agcttggctg
     4561 ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
     4621 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc
     4681 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt
     4741 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     4801 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     4861 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     4921 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
     4981 ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaccaa aatcccttaa
     5041 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttc
//
