LOCUS       sequence-360739-        5410 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor cassette plasmid for high-throughput tagging, encoding an 
            mClover3 synthetic exon in frame 2, as well as a blasticidin 
            resistance gene for selection of genomic integrants..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5410)
  AUTHORS   Serebrenik YV, Sansbury SE, Kumar SS, Henao-Mejia J, Shalem O
  TITLE     Efficient and flexible tagging of endogenous genes by 
            homology-independent intron targeting.
  JOURNAL   Genome Res. 2019 Aug;29(8):1322-1328. doi: 10.1101/gr.246413.118. 
            Epub 2019 Jun 25.
  PUBMED    31239279
REFERENCE   2  (bases 1 to 5410)
  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: 
            "10.1101/gr.246413.118"; journalName: "Genome Res"; date: "2019-08";
            volume: "29"; issue: "8"; pages: "1322-1328"
COMMENT     Sequence Label: pMC_mClover3_BSDminus_F2
FEATURES             Location/Qualifiers
     source          1..5410
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     192..211
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     445..462
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    477..617
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(543..562)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     703..725
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1496..1513)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    1534..1567
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             join(1672..1674,1675..2385)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(1714..1735)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2322..2343
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2442..2523
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     2467..2486
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(2640..3038)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      3035..3044
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3064..3275)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             complement(3446..4240)
                     /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(3446..4240)
                     /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(3557..3576)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4167..4186
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(4604..4621)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4774..4860
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4952..4979
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      join(5113..5410,1..291)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
       61 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
      121 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      181 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      241 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      301 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      361 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      421 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
      481 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
      541 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
      601 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
      661 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
      721 agaggttttc accgtcatca ccgaaacgcg cgaggcagca gatcaattcg cgcgcgaagg
      781 cgaagcggca tgcataatgt gcctgtcaaa tggacgaagc agggattctg caaaccctat
      841 gctactccgt caagccgtca attgtctgat tcgttaccaa ttatgacaac ttgacggcta
      901 catcattcac tttttcttca caaccggcac ggaactcgct cgggctggcc ccggtgcatt
      961 ttttaaatac ccgcgagaaa tagagttgat cgtcaaaacc aacattgcga ccgacggtgg
     1021 cgataggcat ccgggtggtg ctcaaaagca gcttcgcctg gctgatacgt tggtcctcgc
     1081 gccagcttaa gacgctaatc cctaactgct ggcggaaaag atgtgacaga cgcgacggcg
     1141 acaagcaaac atgctgtgcg acgctggcga ttcttctctc atccgccaaa acagccaagc
     1201 tggagaccgt ttgacattac cctgttatcc ctagatacat taccctgtta tcccagatga
     1261 cataccctgt tatccctaga tgacattacc ctgttatccc agatgacatt accctgttat
     1321 ccctagatac attaccctgt tatcccagat gacataccct gttatcccta gatgacatta
     1381 ccctgttatc ccagatgaca ttaccctgtt atccctagat acattaccct gttatcccag
     1441 atgacatacc ctgttatccc tagatgacat taccctgtta tcccagataa actcaatgat
     1501 gatgatgatg atggtcgaga ctcagcggcc gcggtgccag ggcgtgccct tgggctcccc
     1561 gggcgcgact agtgcctcac gaaatcgatt cgctcttcta atctcctctc ttctcctctc
     1621 tccagtggtg gctctggaag ttcaggtgga ggctcgggtg gcggcagttc ggtgagcaag
     1681 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     1741 ggccacaagt tcagcgtccg cggcgagggc gagggcgatg ccaccaacgg caagctgacc
     1801 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     1861 ttcggctacg gcgtggcctg cttcagccgc taccccgacc acatgaagca gcacgacttc
     1921 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatctcttt caaggacgac
     1981 ggtacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2041 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2101 aacttcaaca gccactacgt ctatatcacg gccgacaagc agaagaactg catcaaggct
     2161 aacttcaaga tccgccacaa cgttgaggac ggcagcgtgc agctcgccga ccactaccag
     2221 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagccat
     2281 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2341 gtgaccgccg ccgggattac acatggcatg gacgagctgt acaagggatc cggaagtggc
     2401 tcaagcggag gaggaagtag tggaagttcg taagtattgg tgcagtgaaa aaaatgcttt
     2461 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     2521 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     2581 ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatcc tctggagatt
     2641 tagccctccc acacataacc agagggcagc aattcacgaa tcccaactgc cgtcggctgt
     2701 ccatcactgt ccttcactat ggctttgatc ccaggatgca gatcgagaag cacctgtcgg
     2761 caccgtccgc aggggctcaa gatgcccctg ttctcatttc cgatcgcgac gatacaagtc
     2821 aggttgccag ctgccgcagc agcagcagtg cccagcacca cgagttctgc acaaggtccc
     2881 ccagtaaaat gatatacatt gacaccagtg aagatgcggc cgtcgctaga gagagctgcg
     2941 ctggcgacgc tgtagtcttc agagatgggg atgctgttga ttgtagccgt tgctctttca
     3001 atgagggtgg attcttcttg agacaaaggc ttggccatgg tggcggatcc cgcgtcacga
     3061 cacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga ctactgcact
     3121 tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga catcactttc
     3181 ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc ctccccccaa
     3241 cttctcgggg actgtgggcg atgtgcgctc tgcccactga cgggcaccgg agcctaagag
     3301 cgaatcgatt tcgtgaggcg ccccaactgg ggtaaccttt gagttctctc agttgggggt
     3361 aatcagcatc atgatgtggt accacatcat gatgctgatt ataagaatgc ggccgccaca
     3421 ctctagtgga tctcgagtta ataattcaga agaactcgtc aagaaggcga tagaaggcga
     3481 tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc
     3541 cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtccgcca
     3601 cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc atgatattcg
     3661 gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatg ctcgccttga
     3721 gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga tcatcctgat
     3781 cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc gcttggtggt
     3841 cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca gccatgatgg
     3901 atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc acttcgccca
     3961 atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg caaggaacgc
     4021 ccgtcgtggc cagccacgat agccgcgctg cctcgtcttg cagttcattc agggcaccgg
     4081 acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg aacacggcgg
     4141 catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc tccacccaag
     4201 cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat cctcatcctg
     4261 tctcttgatc agagcttgat cccctgcgcc atcagatcct tggcggcaag aaagccatcc
     4321 agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccggtt
     4381 cgcttgctgt ccataaaacc gcccagtcta gctatcgcca tgtaagccca ctgcaagcta
     4441 cctgctttct ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca
     4501 tccggggtca gcaccgtttc tgcggactgg ctttctacgc gctcgagggg gggccaaacg
     4561 gtctccagct tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat
     4621 cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc
     4681 cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt
     4741 ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa
     4801 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     4861 ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc
     4921 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt
     4981 gcgtttctac aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5041 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     5101 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     5161 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5221 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     5281 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5341 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5401 gttaccggat
//
