LOCUS       sequence-232697-        4117 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4117)
  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 4117)
  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-P33
FEATURES             Location/Qualifiers
     source          1..4117
                     /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..104
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    114..235
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(151..170)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     205..224
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(242..275)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        282..598
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      449..584
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1220..1444
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1451..1699
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1451..1471
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1622..1641
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1701..1734)
                     /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(1771..1790)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1772..1790)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1877..1894)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2048..2636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2128..2147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2807..3598,3599..3667))
                     /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     3430..3449
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3668..3772)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3840..3858
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3896..3918)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4018..4037
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gattacaagg atgacgatga caagtagact agtaacttgt
      121 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
      181 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttagtcga
      241 tataacttcg tatagcatac attatacgaa gttatgtcga cggtgtggaa agtccccagg
      301 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
      361 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
      421 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
      481 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
      541 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
      601 attcgccacc atgactgagt acaagcctac tgtgcgttta gccaccaggg atgatgtgcc
      661 acgagccgtg aggacactag cagccgcctt tgctgactac ccagctacaa ggcacaccgt
      721 ggaccctgac agacacattg aaagagtcac agaacttcag gagctgtttt taactagggt
      781 gggcctggat attggcaaag tctgggtggc cgatgatgga gcagcagttg cagtgtggac
      841 caccccagag tctgttgaag ctggagctgt ctttgcagaa attggaccaa gaatggccga
      901 gttatctggc agcaggctgg ctgctcaaca gcagatggaa ggattattag ctccccacag
      961 acccaaggag cctgcctggt tcctggcaac agttggagtt tctccagacc atcagggcaa
     1021 gggcttagga tctgctgtgg tgctgcctgg tgtggaagca gctgagagag ctggggtgcc
     1081 agcatttctg gagacatcag ctcctagaaa cctgcccttc tatgaacggc tgggcttcac
     1141 agtgactgct gatgtggagg tgcctgaggg ccccagaacc tggtgcatga caagaaaacc
     1201 aggagcctga ctgcagcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     1261 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     1321 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     1381 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     1441 atggctcgag gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc
     1501 tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac
     1561 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat
     1621 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt
     1681 gtggaaagga cgaaacaccg ataacttcgt atagcataca ttatacgaag ttatggtacc
     1741 gatgcagcta gcccgcggat ctgccggtct ccctatagtg agtcgtatta atttcgataa
     1801 gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1861 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     1921 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     1981 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2041 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2101 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2161 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2221 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2281 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2341 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2401 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2461 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2521 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2581 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     2641 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2701 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2761 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2821 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     2881 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     2941 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3001 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3061 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3121 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3181 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3241 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3301 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3361 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3421 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3481 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3541 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3601 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3661 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3721 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     3781 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa
     3841 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct
     3901 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3961 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg
     4021 catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta gaacgcggct
     4081 acaattaata cataacctta tgtatcatac acatacg
//
