LOCUS       sequence-232602-        3863 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mAID-Clover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3863)
  AUTHORS   Fueller J, Herbst K, Meurer M, Gubicza K, Kurtulmus B, Knopf JD, 
            Kirrmaier D, Buchmuller BC, Pereira G, Lemberg MK, Knop M
  TITLE     CRISPR-Cas12a-assisted PCR tagging of mammalian genes.
  JOURNAL   J Cell Biol. 2020 Jun 1;219(6). pii: 151766. doi: 
            10.1083/jcb.201910210.
  PUBMED    32406907
REFERENCE   2  (bases 1 to 3863)
  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"; journalName: "J Cell 
            Biol."; date: "2020-06-1"; volume: "219(6). pii: 151766. doi"; 
            pages: " 10.1083/jcb.201910210"
COMMENT     Sequence Label: pMaCTag-26
FEATURES             Location/Qualifiers
     source          1..3863
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             93..296
                     /codon_start=1
                     /product="minimal degron from the auxin-responsive IAA17 
                     protein of Arabidopsis thaliana (Kubota et al., 2013)"
                     /label=mini-AID
                     /note="acts as an auxin-inducible degron (AID) in the 
                     presence of TIR1"
                     /translation="KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEA
                     AAFVKVSMDGAPYLRKIDLRMYK"
     CDS             join(303..305,306..308,309..1019)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(348..369)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     956..977
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1029..1150
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1066..1085)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1120..1139
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1157..1190)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1197..1445
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1197..1217
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1368..1387
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1447..1480)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1517..1536)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1518..1536)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1623..1640)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1794..2382)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1874..1893)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2553..3344,3345..3413))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3176..3195
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3414..3518)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3586..3604
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3642..3664)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3764..3783
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc ggtgcaggcg ccaaggagaa gagtgcttgt cctaaagatc
      121 cagccaaacc tccggccaag gcacaagttg tgggatggcc accggtgaga tcataccgga
      181 agaacgtgat ggtttcctgc caaaaatcaa gcggtggccc ggaggcggcg gcgttcgtga
      241 aggtatcaat ggacggagca ccgtacttga ggaaaatcga tttgaggatg tataaagcta
      301 gcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      361 acggcgacgt aaacggccac aagttcagcg tccgcggcga gggcgagggc gatgccacca
      421 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
      481 ccctcgtgac caccttcggc tacggcgtgg cctgcttcag ccgctacccc gaccacatga
      541 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
      601 ctttcaagga cgacggtacc tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
      661 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
      721 acaagctgga gtacaacttc aacagccaca acgtctatat cacggccgac aagcagaaga
      781 acggcatcaa ggctaacttc aagatccgcc acaacgttga ggacggcagc gtgcagctcg
      841 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
      901 actacctgag ccatcagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
      961 tcctgctgga gttcgtgacc gccgccggga ttacacatgg catggacgag ctgtacaagt
     1021 gaactagtaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     1081 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     1141 atgtatctta gtcgatataa cttcgtatag catacattat acgaagttat gtcgaggagg
     1201 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     1261 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     1321 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     1381 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     1441 acaccgataa cttcgtatag catacattat acgaagttat ggtaccgatg cagctagccc
     1501 gcggatctgc cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg
     1561 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1621 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1681 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1741 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1801 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1861 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1921 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1981 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2041 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2101 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2161 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2221 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2281 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2341 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2401 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2461 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2521 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2581 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2641 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2701 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     2761 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     2821 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     2881 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     2941 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3001 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3061 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3121 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3181 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3241 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3301 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3361 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3421 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3481 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3541 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     3601 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     3661 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     3721 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     3781 gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata
     3841 accttatgta tcatacacat acg
//
