LOCUS       sequence-228145-        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"
     CDS             64..90
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    100..221
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(137..156)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     191..210
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(228..261)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        268..584
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      435..570
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      591..600
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             597..971
                     /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    981..1205
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1212..1460
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1212..1232
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1383..1402
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1462..1495)
                     /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(1532..1551)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1533..1551)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1638..1655)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1809..2397)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1889..1908)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2568..3359,3360..3428))
                     /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     3191..3210
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3429..3533)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3601..3619
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3657..3679)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3779..3798
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3879..3895,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3879..3895,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tcctacccat acgacgtccc agactacgct tgaactagta acttgtttat tgcagcttat
      121 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
      181 cattctagtt gtggtttgtc caaactcatc aatgtatctt agtcgatata acttcgtata
      241 gcatacatta tacgaagtta tgtcgacggt gtggaaagtc cccaggctcc ccagcaggca
      301 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
      361 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
      421 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
      481 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
      541 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagaattc gccaccatgg
      601 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt
      661 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg
      721 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca
      781 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg
      841 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt
      901 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg
      961 agcaggactg actgcagcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     1021 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     1081 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     1141 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     1201 tatggctcga ggagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     1261 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     1321 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     1381 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     1441 tgtggaaagg acgaaacacc gataacttcg tatagcatac attatacgaa gttatggtac
     1501 cgatgcagct agcccgcgga tctgccggtc tccctatagt gagtcgtatt aatttcgata
     1561 agccaggtta acctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1621 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     1681 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1741 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     1801 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1861 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1921 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1981 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
     2041 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     2101 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     2161 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     2221 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     2281 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2341 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2401 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2461 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     2521 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     2581 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     2641 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     2701 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     2761 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     2821 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     2881 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     2941 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     3001 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     3061 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     3121 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     3181 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     3241 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     3301 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     3361 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     3421 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     3481 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     3541 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa
     3601 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc
     3661 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga
     3721 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg
     3781 gcatcagagc agattgtact gagagtgcac catatggaca tattgtcgtt agaacgcggc
     3841 tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca ctata
//
