LOCUS       sequence-228022-        2990 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with 3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2990)
  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 2990)
  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-34
FEATURES             Location/Qualifiers
     source          1..2990
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..129
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    139..260
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(176..195)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     230..249
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(267..300)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        307..555
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     307..327
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     478..497
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(557..590)
                     /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(627..646)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(628..646)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(733..750)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(904..1492)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(984..1003)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1663..2454,2455..2523))
                     /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     2286..2305
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2524..2628)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2696..2714
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2752..2774)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2874..2893
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(2974..2990,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(2974..2990,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgactaca aagaccatga cggtgattat aaagatcatg atatcgatta caaggatgac
      121 gatgacaagt gaactagtaa cttgtttatt gcagcttata atggttacaa ataaagcaat
      181 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
      241 aaactcatca atgtatctta gtcgatataa cttcgtatag catacattat acgaagttat
      301 gtcgaggagg gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt
      361 agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga
      421 cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac
      481 tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg
      541 aaaggacgaa acaccgataa cttcgtatag catacattat acgaagttat ggtaccgatg
      601 cagctagccc gcggatctgc cggtctccct atagtgagtc gtattaattt cgataagcca
      661 ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
      721 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
      781 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
      841 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
      901 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
      961 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     1021 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     1081 aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     1141 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     1201 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     1261 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     1321 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     1381 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     1441 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     1501 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     1561 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     1621 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     1681 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     1741 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     1801 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     1861 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     1921 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     1981 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     2041 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     2101 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     2161 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     2221 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     2281 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     2341 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     2401 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     2461 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     2521 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     2581 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     2641 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     2701 gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     2761 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     2821 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc
     2881 agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa
     2941 ttaatacata accttatgta tcatacacat acgatttagg tgacactata
//
