LOCUS       sequence-228125-        3892 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3892)
  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 3892)
  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-Z33
FEATURES             Location/Qualifiers
     source          1..3892
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..87
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    97..218
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(134..153)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     188..207
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(225..258)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        265..581
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      432..567
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      588..597
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             594..968
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    978..1202
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1209..1457
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1209..1229
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1380..1399
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1459..1492)
                     /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(1529..1548)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1530..1548)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1635..1652)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1806..2394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1886..1905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2565..3356,3357..3425))
                     /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     3188..3207
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3426..3530)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3598..3616
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3654..3676)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3776..3795
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3876..3892,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3876..3892,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgattaca aggatgacga tgacaagtag actagtaact tgtttattgc agcttataat
      121 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
      181 tctagttgtg gtttgtccaa actcatcaat gtatcttagt cgatataact tcgtatagca
      241 tacattatac gaagttatgt cgacggtgtg gaaagtcccc aggctcccca gcaggcagaa
      301 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
      361 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
      421 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
      481 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
      541 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agaattcgcc accatggcca
      601 agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct
      661 ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc
      721 gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc
      781 tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt
      841 ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg
      901 ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc
      961 aggactgact gcagcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     1021 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     1081 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     1141 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     1201 ggctcgagga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
     1261 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     1321 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     1381 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     1441 ggaaaggacg aaacaccgat aacttcgtat agcatacatt atacgaagtt atggtaccga
     1501 tgcagctagc ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc
     1561 caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1621 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1681 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1741 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1801 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1861 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1921 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1981 ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     2041 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     2101 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2161 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2221 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     2281 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2341 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2401 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2461 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2521 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2581 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2641 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2701 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2761 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2821 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2881 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2941 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     3001 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     3061 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3121 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3181 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3241 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3301 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3361 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3421 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3481 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3541 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     3601 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     3661 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     3721 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     3781 tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac
     3841 aattaataca taaccttatg tatcatacac atacgattta ggtgacacta ta
//
