LOCUS       sequence-232679-        4807 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with mAID-Clover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4807)
  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 4807)
  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-Z26
FEATURES             Location/Qualifiers
     source          1..4807
                     /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..1513
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1364..1499
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1520..1529
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1526..1900
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    1910..2134
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2141..2389
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2141..2161
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2312..2331
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2391..2424)
                     /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(2461..2480)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2462..2480)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2567..2584)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2738..3326)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2818..2837)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3497..4288,4289..4357))
                     /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     4120..4139
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4358..4462)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4530..4548
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4586..4608)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4708..4727
                     /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 aatctagtaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     1081 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     1141 atgtatctta gtcgatataa cttcgtatag catacattat acgaagttat gtcgacggtg
     1201 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     1261 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     1321 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     1381 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     1441 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     1501 aggcttttgc aaagaattcg ccaccatggc caagttgacc agtgccgttc cggtgctcac
     1561 cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt tctcccggga
     1621 cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc
     1681 ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc gcggcctgga
     1741 cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg cctccgggcc
     1801 ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc gcgacccggc
     1861 cggcaactgc gtgcacttcg tggccgagga gcaggactga ctgcagcgac tgtgccttct
     1921 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     1981 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2041 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat
     2101 agcaggcatg ctggggatgc ggtgggctct atggctcgag gagggcctat ttcccatgat
     2161 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac
     2221 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta
     2281 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa
     2341 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg ataacttcgt
     2401 atagcataca ttatacgaag ttatggtacc gatgcagcta gcccgcggat ctgccggtct
     2461 ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc
     2521 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     2581 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     2641 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     2701 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     2761 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     2821 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2881 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2941 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     3001 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     3061 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     3121 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     3181 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     3241 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     3301 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     3361 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     3421 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     3481 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     3541 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     3601 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     3661 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     3721 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     3781 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3841 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3901 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3961 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     4021 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     4081 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     4141 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     4201 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     4261 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     4321 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     4381 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     4441 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     4501 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg
     4561 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag
     4621 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg
     4681 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     4741 atatggacat attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac
     4801 acatacg
//
