LOCUS       sequence-232597-        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"
     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             81..146
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    156..277
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(193..212)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     247..266
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(284..317)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        324..572
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     324..344
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     495..514
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(574..607)
                     /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(644..663)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(645..663)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(750..767)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(921..1509)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1001..1020)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1680..2471,2472..2540))
                     /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     2303..2322
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2541..2645)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2713..2731
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2769..2791)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2891..2910
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gactacaaag accatgacgg tgattataaa gatcatgata
      121 tcgattacaa ggatgacgat gacaagtaga ctagtaactt gtttattgca gcttataatg
      181 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
      241 ctagttgtgg tttgtccaaa ctcatcaatg tatcttagtc gatataactt cgtatagcat
      301 acattatacg aagttatgtc gaggagggcc tatttcccat gattccttca tatttgcata
      361 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
      421 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
      481 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg
      541 ctttatatat cttgtggaaa ggacgaaaca ccgataactt cgtatagcat acattatacg
      601 aagttatggt accgatgcag ctagcccgcg gatctgccgg tctccctata gtgagtcgta
      661 ttaatttcga taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc
      721 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
      781 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
      841 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
      901 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
      961 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1021 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1081 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1141 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1201 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1261 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1321 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     1381 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1441 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1501 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1561 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1621 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     1681 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     1741 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     1801 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     1861 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     1921 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     1981 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     2041 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     2101 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     2161 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     2221 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     2281 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     2341 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     2401 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     2461 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     2521 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     2581 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     2641 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     2701 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac
     2761 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat
     2821 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg
     2881 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg
     2941 ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg
//
