LOCUS       sequence-228073-        5032 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mAID-Clover.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5032)
  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 5032)
  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-P26
FEATURES             Location/Qualifiers
     source          1..5032
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             76..279
                     /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(286..288,289..291,292..1002)
                     /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(331..352)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     939..960
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1012..1133
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1049..1068)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1103..1122
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(1140..1173)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1180..1496
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1347..1482
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2118..2342
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2349..2597
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2349..2369
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2520..2539
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2599..2632)
                     /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(2669..2688)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2670..2688)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2775..2792)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2946..3534)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3026..3045)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3705..4496,4497..4565))
                     /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     4328..4347
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4566..4670)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4738..4756
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4794..4816)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4916..4935
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(5016..5032,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(5016..5032,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccggtgcag gcgccaagga gaagagtgct tgtcctaaag atccagccaa acctccggcc
      121 aaggcacaag ttgtgggatg gccaccggtg agatcatacc ggaagaacgt gatggtttcc
      181 tgccaaaaat caagcggtgg cccggaggcg gcggcgttcg tgaaggtatc aatggacgga
      241 gcaccgtact tgaggaaaat cgatttgagg atgtataaag ctagcatggt gagcaagggc
      301 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
      361 cacaagttca gcgtccgcgg cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg
      421 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc
      481 ggctacggcg tggcctgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
      541 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tctctttcaa ggacgacggt
      601 acctacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
      661 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
      721 ttcaacagcc acaacgtcta tatcacggcc gacaagcaga agaacggcat caaggctaac
      781 ttcaagatcc gccacaacgt tgaggacggc agcgtgcagc tcgccgacca ctaccagcag
      841 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagccatcag
      901 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
      961 accgccgccg ggattacaca tggcatggac gagctgtaca agtgaactag taacttgttt
     1021 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     1081 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttagtcgata
     1141 taacttcgta tagcatacat tatacgaagt tatgtcgacg gtgtggaaag tccccaggct
     1201 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     1261 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     1321 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     1381 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct
     1441 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagaat
     1501 tcgccaccat gactgagtac aagcctactg tgcgtttagc caccagggat gatgtgccac
     1561 gagccgtgag gacactagca gccgcctttg ctgactaccc agctacaagg cacaccgtgg
     1621 accctgacag acacattgaa agagtcacag aacttcagga gctgttttta actagggtgg
     1681 gcctggatat tggcaaagtc tgggtggccg atgatggagc agcagttgca gtgtggacca
     1741 ccccagagtc tgttgaagct ggagctgtct ttgcagaaat tggaccaaga atggccgagt
     1801 tatctggcag caggctggct gctcaacagc agatggaagg attattagct ccccacagac
     1861 ccaaggagcc tgcctggttc ctggcaacag ttggagtttc tccagaccat cagggcaagg
     1921 gcttaggatc tgctgtggtg ctgcctggtg tggaagcagc tgagagagct ggggtgccag
     1981 catttctgga gacatcagct cctagaaacc tgcccttcta tgaacggctg ggcttcacag
     2041 tgactgctga tgtggaggtg cctgagggcc ccagaacctg gtgcatgaca agaaaaccag
     2101 gagcctgact gcagcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     2161 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     2221 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     2281 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     2341 ggctcgagga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     2401 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     2461 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     2521 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     2581 ggaaaggacg aaacaccgat aacttcgtat agcatacatt atacgaagtt atggtaccga
     2641 tgcagctagc ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc
     2701 caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     2761 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     2821 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     2881 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     2941 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3001 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3061 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3121 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3181 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3241 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3301 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3361 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     3421 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     3481 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     3541 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     3601 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3661 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3721 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3781 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3841 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3901 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3961 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4021 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4081 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4141 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4201 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4261 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4321 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4381 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4441 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4501 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4561 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     4621 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     4681 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     4741 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     4801 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     4861 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     4921 tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac
     4981 aattaataca taaccttatg tatcatacac atacgattta ggtgacacta ta
//
