LOCUS       sequence-228074-        4120 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with 1xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4120)
  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 4120)
  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-P27
FEATURES             Location/Qualifiers
     source          1..4120
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..90
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    100..221
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(137..156)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     191..210
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(228..261)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        268..584
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      435..570
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1206..1430
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1437..1685
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1437..1457
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1608..1627
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1687..1720)
                     /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(1757..1776)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1758..1776)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1863..1880)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2034..2622)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2114..2133)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2793..3584,3585..3653))
                     /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     3416..3435
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3654..3758)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3826..3844
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3882..3904)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4004..4023
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4104..4120,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4104..4120,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tcctacccat acgacgtccc agactacgct tgaactagta acttgtttat tgcagcttat
      121 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
      181 cattctagtt gtggtttgtc caaactcatc aatgtatctt agtcgatata acttcgtata
      241 gcatacatta tacgaagtta tgtcgacggt gtggaaagtc cccaggctcc ccagcaggca
      301 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
      361 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
      421 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
      481 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
      541 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagaattc gccaccatga
      601 ctgagtacaa gcctactgtg cgtttagcca ccagggatga tgtgccacga gccgtgagga
      661 cactagcagc cgcctttgct gactacccag ctacaaggca caccgtggac cctgacagac
      721 acattgaaag agtcacagaa cttcaggagc tgtttttaac tagggtgggc ctggatattg
      781 gcaaagtctg ggtggccgat gatggagcag cagttgcagt gtggaccacc ccagagtctg
      841 ttgaagctgg agctgtcttt gcagaaattg gaccaagaat ggccgagtta tctggcagca
      901 ggctggctgc tcaacagcag atggaaggat tattagctcc ccacagaccc aaggagcctg
      961 cctggttcct ggcaacagtt ggagtttctc cagaccatca gggcaagggc ttaggatctg
     1021 ctgtggtgct gcctggtgtg gaagcagctg agagagctgg ggtgccagca tttctggaga
     1081 catcagctcc tagaaacctg cccttctatg aacggctggg cttcacagtg actgctgatg
     1141 tggaggtgcc tgagggcccc agaacctggt gcatgacaag aaaaccagga gcctgactgc
     1201 agcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     1261 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     1321 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     1381 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg ctcgaggagg
     1441 gcctatttcc catgattcct tcatatttgc atatacgata caaggctgtt agagagataa
     1501 ttggaattaa tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt
     1561 aataatttct tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc
     1621 ttaccgtaac ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa
     1681 acaccgataa cttcgtatag catacattat acgaagttat ggtaccgatg cagctagccc
     1741 gcggatctgc cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg
     1801 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1861 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1921 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1981 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2041 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2101 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2161 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2221 ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2281 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     2341 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     2401 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     2461 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     2521 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     2581 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     2641 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     2701 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     2761 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     2821 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     2881 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     2941 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3001 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3061 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3121 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3181 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3241 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3301 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     3361 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     3421 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     3481 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     3541 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     3601 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     3661 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     3721 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3781 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     3841 aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     3901 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     3961 gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt
     4021 gtactgagag tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata
     4081 accttatgta tcatacacat acgatttagg tgacactata
//
