LOCUS       sequence-360737-        5413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor cassette plasmid for high-throughput tagging, encoding an 
            mClover3 synthetic exon in frame 1, 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 5413)
  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 5413)
  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_F1
FEATURES             Location/Qualifiers
     source          1..5413
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     324..345
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    446..527
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     471..490
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(644..1042)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      1039..1048
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1068..1279)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             complement(1450..2244)
                     /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(1450..2244)
                     /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(1561..1580)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2171..2190
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     complement(2608..2625)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      2778..2864
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2956..2983
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     rep_origin      3117..3705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3606..3625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3859..3876
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3891..4031
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3957..3976)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4117..4139
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4910..4927)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     protein_bind    4948..4981
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="minimal attB site for the phi-C31 integrase (Groth 
                     et al., 2000)"
     CDS             join(5087..5089,5090..5413,1..387)
                     /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(5129..5150)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
       61 ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttcaa cagccactac
      121 gtctatatca cggccgacaa gcagaagaac tgcatcaagg ctaacttcaa gatccgccac
      181 aacgttgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
      241 gacggccccg tgctgctgcc cgacaaccac tacctgagcc atcagtccaa gctgagcaaa
      301 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatt
      361 acacatggca tggacgagct gtacaaggga tccggaagtg gctcaagcgg aggaggaagt
      421 agtggaagtt ctggtaagta ttggtgcagt gaaaaaaatg ctttatttgt gaaatttgtg
      481 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
      541 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttaa agcaagtaaa
      601 acctctacaa atgtggtatg gctgattatg atcctctgga gatttagccc tcccacacat
      661 aaccagaggg cagcaattca cgaatcccaa ctgccgtcgg ctgtccatca ctgtccttca
      721 ctatggcttt gatcccagga tgcagatcga gaagcacctg tcggcaccgt ccgcaggggc
      781 tcaagatgcc cctgttctca tttccgatcg cgacgataca agtcaggttg ccagctgccg
      841 cagcagcagc agtgcccagc accacgagtt ctgcacaagg tcccccagta aaatgatata
      901 cattgacacc agtgaagatg cggccgtcgc tagagagagc tgcgctggcg acgctgtagt
      961 cttcagagat ggggatgctg ttgattgtag ccgttgctct ttcaatgagg gtggattctt
     1021 cttgagacaa aggcttggcc atggtggcgg atcccgcgtc acgacacctg tgttctggcg
     1081 gcaaacccgt tgcgaaaaag aacgttcacg gcgactactg cacttatata cggttctccc
     1141 ccaccctcgg gaaaaaggcg gagccagtac acgacatcac tttcccagtt taccccgcgc
     1201 caccttctct aggcaccggt tcaattgccg acccctcccc ccaacttctc ggggactgtg
     1261 ggcgatgtgc gctctgccca ctgacgggca ccggagccta agagcgaatc gatttcgtga
     1321 ggcgccccaa ctggggtaac ctttgagttc tctcagttgg gggtaatcag catcatgatg
     1381 tggtaccaca tcatgatgct gattataaga atgcggccgc cacactctag tggatctcga
     1441 gttaataatt cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg
     1501 agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc
     1561 aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca
     1621 gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc
     1681 atgggtcacg acgagatcct cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc
     1741 ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc
     1801 catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc
     1861 cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg
     1921 agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct
     1981 tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca
     2041 cgatagccgc gctgcctcgt cttgcagttc attcagggca ccggacaggt cggtcttgac
     2101 aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat
     2161 tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc
     2221 gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagagct
     2281 tgatcccctg cgccatcaga tccttggcgg caagaaagcc atccagttta ctttgcaggg
     2341 cttcccaacc ttaccagagg gcgccccagc tggcaattcc ggttcgcttg ctgtccataa
     2401 aaccgcccag tctagctatc gccatgtaag cccactgcaa gctacctgct ttctctttgc
     2461 gcttgcgttt tcccttgtcc agatagccca gtagctgaca ttcatccggg gtcagcaccg
     2521 tttctgcgga ctggctttct acgcgctcga gggggggcca aacggtctcc agcttggctg
     2581 ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg
     2641 tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc
     2701 gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt
     2761 agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     2821 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt
     2881 tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca
     2941 ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc
     3001 ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaccaa aatcccttaa
     3061 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3121 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3181 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3241 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     3301 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3361 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3421 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3481 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3541 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3601 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3661 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     3721 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     3781 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     3841 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     3901 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca
     3961 atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg
     4021 tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4081 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4141 tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg aaggcgaagc
     4201 ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc ctatgctact
     4261 ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg gctacatcat
     4321 tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa
     4381 atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag
     4441 gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc
     4501 ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc
     4561 aaacatgctg tgcgacgctg gcgattcttc tctcatccgc caaaacagcc aagctggaga
     4621 ccgtttgaca ttaccctgtt atccctagat acattaccct gttatcccag atgacatacc
     4681 ctgttatccc tagatgacat taccctgtta tcccagatga cattaccctg ttatccctag
     4741 atacattacc cygttatccc agatgacata ccctgttatc cctagatgac attaccctgt
     4801 tatcccagat gacattaccc tgttatccct agatacatta ccctgttatc ccagatgaca
     4861 taccctgtta tccctagatg acattaccct gttatcccag ataaactcaa tgatgatgat
     4921 gatgatggtc gagactcagc ggccgcggtg ccagggcgtg cccttgggct ccccgggcgc
     4981 gactagtgcc tcacgaaatc gattcgctct tctaatctcc tctcttctcc tctctccagc
     5041 tggtggctct ggaagttcag gtggaggctc gggtggcggc agttcggtga gcaagggcga
     5101 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     5161 caagttcagc gtccgcggcg agggcgaggg cgatgccacc aacggcaagc tgaccctgaa
     5221 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccttcgg
     5281 ctacggcgtg gcctgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa
     5341 gtccgccatg cccgaaggct acgtccagga gcgcaccatc tctttcaagg acgacggtac
     5401 ctacaagacc cgc
//
