LOCUS       sequence-232645-        4183 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with 3xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4183)
  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 4183)
  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-P28
FEATURES             Location/Qualifiers
     source          1..4183
                     /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..170
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     polyA_signal    180..301
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(217..236)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     271..290
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(308..341)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        348..664
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      515..650
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1286..1510
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1517..1765
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1517..1537
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1688..1707
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1767..1800)
                     /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(1837..1856)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1838..1856)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1943..1960)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2114..2702)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2194..2213)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2873..3664,3665..3733))
                     /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     3496..3515
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3734..3838)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3906..3924
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3962..3984)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4084..4103
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc tacccatacg atgttcctga ctatgcgggc tatccctatg
      121 acgtcccgga ctatgcagga tcctatccat atgacgttcc agattacgct tgaactagta
      181 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
      241 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
      301 agtcgatata acttcgtata gcatacatta tacgaagtta tgtcgacggt gtggaaagtc
      361 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
      421 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
      481 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
      541 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
      601 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
      661 caaagaattc gccaccatga ctgagtacaa gcctactgtg cgtttagcca ccagggatga
      721 tgtgccacga gccgtgagga cactagcagc cgcctttgct gactacccag ctacaaggca
      781 caccgtggac cctgacagac acattgaaag agtcacagaa cttcaggagc tgtttttaac
      841 tagggtgggc ctggatattg gcaaagtctg ggtggccgat gatggagcag cagttgcagt
      901 gtggaccacc ccagagtctg ttgaagctgg agctgtcttt gcagaaattg gaccaagaat
      961 ggccgagtta tctggcagca ggctggctgc tcaacagcag atggaaggat tattagctcc
     1021 ccacagaccc aaggagcctg cctggttcct ggcaacagtt ggagtttctc cagaccatca
     1081 gggcaagggc ttaggatctg ctgtggtgct gcctggtgtg gaagcagctg agagagctgg
     1141 ggtgccagca tttctggaga catcagctcc tagaaacctg cccttctatg aacggctggg
     1201 cttcacagtg actgctgatg tggaggtgcc tgagggcccc agaacctggt gcatgacaag
     1261 aaaaccagga gcctgactgc agcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1321 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1381 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     1441 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     1501 ggctctatgg ctcgaggagg gcctatttcc catgattcct tcatatttgc atatacgata
     1561 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     1621 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     1681 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     1741 tatcttgtgg aaaggacgaa acaccgataa cttcgtatag catacattat acgaagttat
     1801 ggtaccgatg cagctagccc gcggatctgc cggtctccct atagtgagtc gtattaattt
     1861 cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     1921 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     1981 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2041 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2101 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2161 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2221 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2281 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2341 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2401 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2461 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2521 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2581 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2641 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     2701 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     2761 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     2821 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     2881 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     2941 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3001 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3061 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3121 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3181 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3241 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3301 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3361 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3421 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3481 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3541 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3601 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3661 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     3721 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     3781 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     3841 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     3901 ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa
     3961 acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
     4021 gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact
     4081 atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg tcgttagaac
     4141 gcggctacaa ttaatacata accttatgta tcatacacat acg
//
