LOCUS       sequence-232643-        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"
     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"
     polyA_signal    2135..2359
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2366..2614
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2366..2386
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2537..2556
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2616..2649)
                     /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(2686..2705)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2687..2705)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2792..2809)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2963..3551)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3043..3062)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3722..4513,4514..4582))
                     /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     4345..4364
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4583..4687)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4755..4773
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4811..4833)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4933..4952
                     /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 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 ccaccatgac tgagtacaag cctactgtgc gtttagccac
     1561 cagggatgat gtgccacgag ccgtgaggac actagcagcc gcctttgctg actacccagc
     1621 tacaaggcac accgtggacc ctgacagaca cattgaaaga gtcacagaac ttcaggagct
     1681 gtttttaact agggtgggcc tggatattgg caaagtctgg gtggccgatg atggagcagc
     1741 agttgcagtg tggaccaccc cagagtctgt tgaagctgga gctgtctttg cagaaattgg
     1801 accaagaatg gccgagttat ctggcagcag gctggctgct caacagcaga tggaaggatt
     1861 attagctccc cacagaccca aggagcctgc ctggttcctg gcaacagttg gagtttctcc
     1921 agaccatcag ggcaagggct taggatctgc tgtggtgctg cctggtgtgg aagcagctga
     1981 gagagctggg gtgccagcat ttctggagac atcagctcct agaaacctgc ccttctatga
     2041 acggctgggc ttcacagtga ctgctgatgt ggaggtgcct gagggcccca gaacctggtg
     2101 catgacaaga aaaccaggag cctgactgca gcgactgtgc cttctagttg ccagccatct
     2161 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     2221 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     2281 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     2341 gatgcggtgg gctctatggc tcgaggaggg cctatttccc atgattcctt catatttgca
     2401 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     2461 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     2521 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     2581 ggctttatat atcttgtgga aaggacgaaa caccgataac ttcgtatagc atacattata
     2641 cgaagttatg gtaccgatgc agctagcccg cggatctgcc ggtctcccta tagtgagtcg
     2701 tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag
     2761 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     2821 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2881 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2941 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     3001 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3061 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3121 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3181 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3241 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3301 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3361 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3421 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3481 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3541 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3601 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3661 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3721 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     3781 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3841 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     3901 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     3961 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     4021 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     4081 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4141 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4201 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4261 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4321 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4381 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4441 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4501 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4561 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4621 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4681 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     4741 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     4801 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4861 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     4921 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt
     4981 cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cg
//
