LOCUS       sequence-232686-        3913 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with S-Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3913)
  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 3913)
  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-Z32
FEATURES             Location/Qualifiers
     source          1..3913
                     /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..125
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     polyA_signal    135..256
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(172..191)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     226..245
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(263..296)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        303..619
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      470..605
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      626..635
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             632..1006
                     /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    1016..1240
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1247..1495
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1247..1267
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1418..1437
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1497..1530)
                     /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(1567..1586)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1568..1586)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1673..1690)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1844..2432)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1924..1943)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2603..3394,3395..3463))
                     /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     3226..3245
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3464..3568)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3636..3654
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3692..3714)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3814..3833
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc aaagaaaccg ctgctgctaa attcgaacgc cagcacatgg
      121 acagctgaac tagtaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
      181 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
      241 tcatcaatgt atcttagtcg atataacttc gtatagcata cattatacga agttatgtcg
      301 acggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
      361 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
      421 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
      481 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
      541 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
      601 aggcctaggc ttttgcaaag aattcgccac catggccaag ttgaccagtg ccgttccggt
      661 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
      721 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
      781 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
      841 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
      901 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
      961 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgactgc agcgactgtg
     1021 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1081 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1141 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1201 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg ctcgaggagg gcctatttcc
     1261 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa
     1321 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     1381 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     1441 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgataa
     1501 cttcgtatag catacattat acgaagttat ggtaccgatg cagctagccc gcggatctgc
     1561 cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa
     1621 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     1681 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     1741 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     1801 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     1861 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     1921 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     1981 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2041 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2101 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2161 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     2221 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     2281 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     2341 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     2401 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     2461 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     2521 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     2581 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     2641 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     2701 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     2761 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     2821 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     2881 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     2941 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3001 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3061 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3121 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3181 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     3241 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     3301 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     3361 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     3421 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     3481 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     3541 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     3601 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     3661 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg
     3721 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg
     3781 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag
     3841 tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta
     3901 tcatacacat acg
//
