LOCUS       sequence-232680-        3895 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with 1xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3895)
  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 3895)
  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-Z27
FEATURES             Location/Qualifiers
     source          1..3895
                     /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..107
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    117..238
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(154..173)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     208..227
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(245..278)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        285..601
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      452..587
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      608..617
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             614..988
                     /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    998..1222
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1229..1477
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1229..1249
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1400..1419
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1479..1512)
                     /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(1549..1568)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1550..1568)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1655..1672)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1826..2414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1906..1925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2585..3376,3377..3445))
                     /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     3208..3227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3446..3550)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3618..3636
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3674..3696)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3796..3815
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc tacccatacg acgtcccaga ctacgcttga actagtaact
      121 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
      181 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttagt
      241 cgatataact tcgtatagca tacattatac gaagttatgt cgacggtgtg gaaagtcccc
      301 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
      361 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      421 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
      481 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
      541 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
      601 agaattcgcc accatggcca agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt
      661 cgccggagcg gtcgagttct ggaccgaccg gctcgggttc tcccgggact tcgtggagga
      721 cgacttcgcc ggtgtggtcc gggacgacgt gaccctgttc atcagcgcgg tccaggacca
      781 ggtggtgccg gacaacaccc tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc
      841 cgagtggtcg gaggtcgtgt ccacgaactt ccgggacgcc tccgggccgg ccatgaccga
      901 gatcggcgag cagccgtggg ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt
      961 gcacttcgtg gccgaggagc aggactgact gcagcgactg tgccttctag ttgccagcca
     1021 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     1081 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     1141 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     1201 ggggatgcgg tgggctctat ggctcgagga gggcctattt cccatgattc cttcatattt
     1261 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa
     1321 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt
     1381 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt
     1441 cttggcttta tatatcttgt ggaaaggacg aaacaccgat aacttcgtat agcatacatt
     1501 atacgaagtt atggtaccga tgcagctagc ccgcggatct gccggtctcc ctatagtgag
     1561 tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga
     1621 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     1681 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     1741 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1801 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1861 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1921 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1981 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     2041 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     2101 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     2161 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     2221 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     2281 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     2341 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     2401 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     2461 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2521 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2581 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2641 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2701 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     2761 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2821 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2881 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2941 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3001 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     3061 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     3121 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     3181 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     3241 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     3301 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     3361 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     3421 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     3481 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     3541 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     3601 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     3661 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     3721 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     3781 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat
     3841 tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacg
//
