LOCUS       sequence-228031-        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"
     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..1428
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1180..1200
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1351..1370
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1430..1463)
                     /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(1500..1519)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1501..1519)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1606..1623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1777..2365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1857..1876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2536..3327,3328..3396))
                     /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     3159..3178
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3397..3501)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3569..3587
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3625..3647)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3747..3766
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3847..3863,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3847..3863,1)
                     /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 tatgtcgagg agggcctatt tcccatgatt
     1201 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
     1261 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     1321 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     1381 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga taacttcgta
     1441 tagcatacat tatacgaagt tatggtaccg atgcagctag cccgcggatc tgccggtctc
     1501 cctatagtga gtcgtattaa tttcgataag ccaggttaac ctgcattaat gaatcggcca
     1561 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     1621 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1681 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1741 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1801 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1861 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1921 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     1981 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2041 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2101 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2161 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     2221 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2281 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2341 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2401 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2461 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2521 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2581 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2641 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2701 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     2761 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     2821 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2881 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2941 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3001 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3061 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3121 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3181 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3241 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3301 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3361 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3421 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3481 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     3541 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     3601 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     3661 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     3721 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     3781 tatggacata ttgtcgttag aacgcggcta caattaatac ataaccttat gtatcataca
     3841 catacgattt aggtgacact ata
//
