LOCUS       sequence-228149-        3910 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with V5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3910)
  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 3910)
  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-Z31
FEATURES             Location/Qualifiers
     source          1..3910
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..105
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     polyA_signal    115..236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(152..171)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     206..225
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(243..276)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        283..599
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      450..585
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      606..615
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             612..986
                     /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    996..1220
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1227..1475
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1227..1247
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1398..1417
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1477..1510)
                     /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(1547..1566)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1548..1566)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1653..1670)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1824..2412)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1904..1923)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2583..3374,3375..3443))
                     /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     3206..3225
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3444..3548)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3616..3634
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3672..3694)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3794..3813
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3894..3910,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3894..3910,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgtgaac tagtaacttg
      121 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
      181 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttagtcg
      241 atataacttc gtatagcata cattatacga agttatgtcg acggtgtgga aagtccccag
      301 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
      361 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
      421 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
      481 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
      541 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
      601 aattcgccac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
      661 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
      721 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
      781 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
      841 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
      901 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
      961 acttcgtggc cgaggagcag gactgactgc agcgactgtg ccttctagtt gccagccatc
     1021 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1081 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1141 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     1201 ggatgcggtg ggctctatgg ctcgaggagg gcctatttcc catgattcct tcatatttgc
     1261 atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga
     1321 tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa
     1381 aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct
     1441 tggctttata tatcttgtgg aaaggacgaa acaccgataa cttcgtatag catacattat
     1501 acgaagttat ggtaccgatg cagctagccc gcggatctgc cggtctccct atagtgagtc
     1561 gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg cgcggggaga
     1621 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     1681 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     1741 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     1801 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     1861 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     1921 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     1981 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc
     2041 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     2101 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2161 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2221 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     2281 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     2341 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     2401 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     2461 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     2521 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     2581 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     2641 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     2701 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     2761 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     2821 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     2881 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     2941 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     3001 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     3061 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     3121 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     3181 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3241 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3301 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     3361 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     3421 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     3481 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     3541 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     3601 acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat
     3661 gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg
     3721 gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
     3781 tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat ggacatattg
     3841 tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat acgatttagg
     3901 tgacactata
//
