LOCUS       sequence-232681-        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"
     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..170
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     polyA_signal    180..301
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(217..236)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     271..290
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(308..341)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        348..664
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      515..650
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      671..680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             677..1051
                     /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    1061..1285
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1292..1540
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1292..1312
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1463..1482
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1542..1575)
                     /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(1612..1631)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1613..1631)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1718..1735)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1889..2477)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1969..1988)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2648..3439,3440..3508))
                     /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     3271..3290
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3509..3613)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3681..3699
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3737..3759)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3859..3878
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc tacccatacg atgttcctga ctatgcgggc tatccctatg
      121 acgtcccgga ctatgcagga tcctatccat atgacgttcc agattacgct tgaactagta
      181 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
      241 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
      301 agtcgatata acttcgtata gcatacatta tacgaagtta tgtcgacggt gtggaaagtc
      361 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
      421 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
      481 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
      541 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
      601 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
      661 caaagaattc gccaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga
      721 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga
      781 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga
      841 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta
      901 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac
      961 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg
     1021 cgtgcacttc gtggccgagg agcaggactg actgcagcga ctgtgccttc tagttgccag
     1081 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1141 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1201 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     1261 gctggggatg cggtgggctc tatggctcga ggagggccta tttcccatga ttccttcata
     1321 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac
     1381 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
     1441 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
     1501 tttcttggct ttatatatct tgtggaaagg acgaaacacc gataacttcg tatagcatac
     1561 attatacgaa gttatggtac cgatgcagct agcccgcgga tctgccggtc tccctatagt
     1621 gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc caacgcgcgg
     1681 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1741 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1801 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1861 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1921 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1981 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     2041 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     2101 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     2161 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     2221 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     2281 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2341 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2401 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2461 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2521 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2581 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2641 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2701 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2761 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2821 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     2881 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     2941 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     3001 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     3061 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     3121 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     3181 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     3241 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3301 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3361 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3421 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3481 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3541 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3601 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     3661 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     3721 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     3781 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     3841 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatggaca
     3901 tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata cacatacg
//
