LOCUS       sequence-228033-        3014 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with 3xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3014)
  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 3014)
  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-28
FEATURES             Location/Qualifiers
     source          1..3014
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..153
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     polyA_signal    163..284
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(200..219)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     254..273
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(291..324)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        331..579
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     331..351
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     502..521
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(581..614)
                     /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(651..670)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(652..670)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(757..774)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(928..1516)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1008..1027)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1687..2478,2479..2547))
                     /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     2310..2329
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2548..2652)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2720..2738
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2776..2798)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2898..2917
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(2998..3014,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(2998..3014,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tcctacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc ggactatgca
      121 ggatcctatc catatgacgt tccagattac gcttgaacta gtaacttgtt tattgcagct
      181 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
      241 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttagtcgat ataacttcgt
      301 atagcataca ttatacgaag ttatgtcgag gagggcctat ttcccatgat tccttcatat
      361 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
      421 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
      481 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
      541 ttcttggctt tatatatctt gtggaaagga cgaaacaccg ataacttcgt atagcataca
      601 ttatacgaag ttatggtacc gatgcagcta gcccgcggat ctgccggtct ccctatagtg
      661 agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg
      721 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
      781 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
      841 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
      901 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
      961 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     1021 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     1081 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta
     1141 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     1201 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     1261 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     1321 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     1381 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     1441 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     1501 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     1561 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     1621 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     1681 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     1741 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     1801 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     1861 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     1921 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     1981 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     2041 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     2101 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     2161 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     2221 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     2281 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     2341 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     2401 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     2461 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     2521 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     2581 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     2641 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     2701 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     2761 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     2821 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     2881 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat
     2941 attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt
     3001 taggtgacac tata
//
