LOCUS       sequence-232637-        4159 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with 3xFLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4159)
  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 4159)
  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-P34
FEATURES             Location/Qualifiers
     source          1..4159
                     /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..640
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      491..626
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1262..1486
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1493..1741
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1493..1513
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1664..1683
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1743..1776)
                     /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(1813..1832)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1814..1832)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1919..1936)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2090..2678)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2170..2189)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2849..3640,3641..3709))
                     /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     3472..3491
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3710..3814)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3882..3900
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3938..3960)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4060..4079
                     /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 gacggtgtgg aaagtcccca ggctccccag caggcagaag
      361 tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc
      421 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct
      481 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
      541 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
      601 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gaattcgcca ccatgactga
      661 gtacaagcct actgtgcgtt tagccaccag ggatgatgtg ccacgagccg tgaggacact
      721 agcagccgcc tttgctgact acccagctac aaggcacacc gtggaccctg acagacacat
      781 tgaaagagtc acagaacttc aggagctgtt tttaactagg gtgggcctgg atattggcaa
      841 agtctgggtg gccgatgatg gagcagcagt tgcagtgtgg accaccccag agtctgttga
      901 agctggagct gtctttgcag aaattggacc aagaatggcc gagttatctg gcagcaggct
      961 ggctgctcaa cagcagatgg aaggattatt agctccccac agacccaagg agcctgcctg
     1021 gttcctggca acagttggag tttctccaga ccatcagggc aagggcttag gatctgctgt
     1081 ggtgctgcct ggtgtggaag cagctgagag agctggggtg ccagcatttc tggagacatc
     1141 agctcctaga aacctgccct tctatgaacg gctgggcttc acagtgactg ctgatgtgga
     1201 ggtgcctgag ggccccagaa cctggtgcat gacaagaaaa ccaggagcct gactgcagcg
     1261 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1321 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1381 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1441 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctcg aggagggcct
     1501 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
     1561 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
     1621 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
     1681 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
     1741 cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc tagcccgcgg
     1801 atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt aacctgcatt
     1861 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     1921 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     1981 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2041 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2101 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2161 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2221 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     2281 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     2341 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     2401 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     2461 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     2521 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     2581 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     2641 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     2701 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     2761 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     2821 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     2881 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     2941 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     3001 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3061 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3121 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3181 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3241 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     3301 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     3361 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     3421 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     3481 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     3541 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     3601 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     3661 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     3721 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     3781 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     3841 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     3901 cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     3961 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     4021 cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag cagattgtac
     4081 tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa tacataacct
     4141 tatgtatcat acacatacg
//
