LOCUS       sequence-228146-        3958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with 3xHA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3958)
  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 3958)
  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-Z28
FEATURES             Location/Qualifiers
     source          1..3958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..153
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     polyA_signal    163..284
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(200..219)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     254..273
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(291..324)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        331..647
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      498..633
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      654..663
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             660..1034
                     /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    1044..1268
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1275..1523
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1275..1295
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1446..1465
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1525..1558)
                     /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(1595..1614)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1596..1614)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1701..1718)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1872..2460)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1952..1971)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2631..3422,3423..3491))
                     /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     3254..3273
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3492..3596)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3664..3682
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3720..3742)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3842..3861
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3942..3958,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3942..3958,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tcctacccat acgatgttcc tgactatgcg ggctatccct atgacgtccc ggactatgca
      121 ggatcctatc catatgacgt tccagattac gcttgaacta gtaacttgtt tattgcagct
      181 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
      241 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttagtcgat ataacttcgt
      301 atagcataca ttatacgaag ttatgtcgac ggtgtggaaa gtccccaggc tccccagcag
      361 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
      421 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
      481 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
      541 atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat
      601 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaagaa ttcgccacca
      661 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
      721 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
      781 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
      841 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
      901 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
      961 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     1021 aggagcagga ctgactgcag cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     1081 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     1141 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     1201 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     1261 ctctatggct cgaggagggc ctatttccca tgattccttc atatttgcat atacgataca
     1321 aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa
     1381 atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta
     1441 aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata
     1501 tcttgtggaa aggacgaaac accgataact tcgtatagca tacattatac gaagttatgg
     1561 taccgatgca gctagcccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg
     1621 ataagccagg ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     1681 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     1741 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     1801 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     1861 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     1921 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     1981 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     2041 ccttcgggaa gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag
     2101 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     2161 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     2221 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     2281 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     2341 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     2401 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     2461 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     2521 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2581 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2641 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2701 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     2761 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2821 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2881 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     2941 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     3001 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3061 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3121 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3181 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3241 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     3301 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3361 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3421 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3481 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     3541 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     3601 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     3661 aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac
     3721 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     3781 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat
     3841 gcggcatcag agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc
     3901 ggctacaatt aatacataac cttatgtatc atacacatac gatttaggtg acactata
//
