LOCUS       sequence-360742-        5505 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor cassette plasmid for high-throughput tagging, encoding an 
            mClover3 synthetic exon in frame 0, 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 5505)
  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 5505)
  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_F0
FEATURES             Location/Qualifiers
     source          1..5505
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(155..172)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    193..226
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             join(330..332,333..1043)
                     /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(372..393)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     980..1001
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1101..1182
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     1126..1145
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(1299..1697)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      1694..1703
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1723..1934)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             complement(2105..2899)
                     /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(2105..2899)
                     /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(2216..2235)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2826..2845
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(3263..3280)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3433..3519
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3611..3638
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      3772..4360
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4261..4280
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4514..4531
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4546..4686
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4612..4631)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4772..4794
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 atcccagatg acataccctg ttatccctag atgacattac cctgttatcc cagatgacat
       61 taccctgtta tccctagata cattaccctg ttatcccaga tgacataccc tgttatccct
      121 agatgacatt accctgttat cccagataaa ctcaatgatg atgatgatga tggtcgagac
      181 tcagcggccg cggtgccagg gcgtgccctt gggctccccg ggcgcgacta gtgcctcacg
      241 aaatcgattc gctcttctaa tctcctctct tctcctctct ccagggtggc tctggaagtt
      301 caggtggagg ctcgggtggc ggcagttcgg tgagcaaggg cgaggagctg ttcaccgggg
      361 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtccgcg
      421 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
      481 gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt cggctacggc gtggcctgct
      541 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
      601 gctacgtcca ggagcgcacc atctctttca aggacgacgg tacctacaag acccgcgccg
      661 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
      721 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cactacgtct
      781 atatcacggc cgacaagcag aagaactgca tcaaggctaa cttcaagatc cgccacaacg
      841 ttgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
      901 gccccgtgct gctgcccgac aaccactacc tgagccatca gtccaagctg agcaaagacc
      961 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggattacac
     1021 atggcatgga cgagctgtac aagggatccg gaagtggctc aagcggagga ggaagtagtg
     1081 gaagttctgt aagtattggt gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct
     1141 attgctttat ttgtaaccat tataagctgc aataaacaag ttaacaacaa caattgcatt
     1201 cattttatgt ttcaggttca gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc
     1261 tacaaatgtg gtatggctga ttatgatcct ctggagattt agccctccca cacataacca
     1321 gagggcagca attcacgaat cccaactgcc gtcggctgtc catcactgtc cttcactatg
     1381 gctttgatcc caggatgcag atcgagaagc acctgtcggc accgtccgca ggggctcaag
     1441 atgcccctgt tctcatttcc gatcgcgacg atacaagtca ggttgccagc tgccgcagca
     1501 gcagcagtgc ccagcaccac gagttctgca caaggtcccc cagtaaaatg atatacattg
     1561 acaccagtga agatgcggcc gtcgctagag agagctgcgc tggcgacgct gtagtcttca
     1621 gagatgggga tgctgttgat tgtagccgtt gctctttcaa tgagggtgga ttcttcttga
     1681 gacaaaggct tggccatggt ggcggatccc gcgtcacgac acctgtgttc tggcggcaaa
     1741 cccgttgcga aaaagaacgt tcacggcgac tactgcactt atatacggtt ctcccccacc
     1801 ctcgggaaaa aggcggagcc agtacacgac atcactttcc cagtttaccc cgcgccacct
     1861 tctctaggca ccggttcaat tgccgacccc tccccccaac ttctcgggga ctgtgggcga
     1921 tgtgcgctct gcccactgac gggcaccgga gcctaagagc gaatcgattt cgtgaggcgc
     1981 cccaactggg gtaacctttg agttctctca gttgggggta atcagcatca tgatgtggta
     2041 ccacatcatg atgctgatta taagaatgcg gccgccacac tctagtggat ctcgagttaa
     2101 taattcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg
     2161 cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat
     2221 cacgggtagc caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga
     2281 tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg
     2341 tcacgacgag atcctcgccg tcgggcatgc tcgccttgag cctggcgaac agttcggctg
     2401 gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc
     2461 gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat
     2521 caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa
     2581 ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg
     2641 cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata
     2701 gccgcgctgc ctcgtcttgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa
     2761 gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct
     2821 gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca
     2881 atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt ctcttgatca gagcttgatc
     2941 ccctgcgcca tcagatcctt ggcggcaaga aagccatcca gtttactttg cagggcttcc
     3001 caaccttacc agagggcgcc ccagctggca attccggttc gcttgctgtc cataaaaccg
     3061 cccagtctag ctatcgccat gtaagcccac tgcaagctac ctgctttctc tttgcgcttg
     3121 cgttttccct tgtccagata gcccagtagc tgacattcat ccggggtcag caccgtttct
     3181 gcggactggc tttctacgcg ctcgaggggg ggccaaacgg tctccagctt ggctgttttg
     3241 gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga
     3301 taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact
     3361 cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga
     3421 actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     3481 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac
     3541 gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat
     3601 caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttttg
     3661 tttatttttc taaatacatt caaatatgta tccgctcatg accaaaatcc cttaacgtga
     3721 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     3781 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     3841 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     3901 gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     3961 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4021 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4081 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4141 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4201 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4261 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4321 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4381 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     4441 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     4501 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
     4561 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
     4621 ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg
     4681 gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
     4741 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
     4801 ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc gaagcggcat
     4861 gcataatgtg cctgtcaaat ggacgaagca gggattctgc aaaccctatg ctactccgtc
     4921 aagccgtcaa ttgtctgatt cgttaccaat tatgacaact tgacggctac atcattcact
     4981 ttttcttcac aaccggcacg gaactcgctc gggctggccc cggtgcattt tttaaatacc
     5041 cgcgagaaat agagttgatc gtcaaaacca acattgcgac cgacggtggc gataggcatc
     5101 cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag
     5161 acgctaatcc ctaactgctg gcggaaaaga tgtgacagac gcgacggcga caagcaaaca
     5221 tgctgtgcga cgctggcgat tcttctctca tccgccaaaa cagccaagct ggagaccgtt
     5281 tgacattacc ctgttatccc tagatacatt accctgttat cccagatgac ataccctgtt
     5341 atccctagat gacattaccc tgttatccca gatgacatta ccctgttatc cctagataca
     5401 ttaccctgtt atcccagatg acataccctg ttatccctag atgacattac cctgttatcc
     5461 cagatgacat taccctgtta tccctagata cattaccctg ttgtt
//
