LOCUS       sequence-232503-        2948 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2948)
  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 2948)
  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-33
FEATURES             Location/Qualifiers
     source          1..2948
                     /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..530
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     282..302
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     453..472
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(532..565)
                     /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(602..621)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(603..621)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(708..725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(879..1467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(959..978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1638..2429,2430..2498))
                     /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     2261..2280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2499..2603)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2671..2689
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2727..2749)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2849..2868
                     /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 ggagggccta tttcccatga
      301 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga
      361 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
      421 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
      481 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc gataacttcg
      541 tatagcatac attatacgaa gttatggtac cgatgcagct agcccgcgga tctgccggtc
      601 tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc
      661 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
      721 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
      781 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
      841 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
      901 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
      961 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1021 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1081 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1141 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1201 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1261 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     1321 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     1381 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     1441 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     1501 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     1561 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     1621 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     1681 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     1741 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     1801 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     1861 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     1921 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     1981 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     2041 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     2101 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     2161 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     2221 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     2281 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     2341 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     2401 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     2461 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     2521 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     2581 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     2641 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     2701 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     2761 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     2821 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     2881 catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata
     2941 cacatacg
//
