LOCUS       sequence-232655-        3892 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3892)
  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 3892)
  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-Z33
FEATURES             Location/Qualifiers
     source          1..3892
                     /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..104
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    114..235
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(151..170)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     205..224
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(242..275)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        282..598
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      449..584
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      605..614
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             611..985
                     /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    995..1219
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1226..1474
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1226..1246
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1397..1416
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1476..1509)
                     /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(1546..1565)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1547..1565)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1652..1669)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1823..2411)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1903..1922)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2582..3373,3374..3442))
                     /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     3205..3224
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3443..3547)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3615..3633
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3671..3693)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3793..3812
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gattacaagg atgacgatga caagtagact agtaacttgt
      121 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
      181 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttagtcga
      241 tataacttcg tatagcatac attatacgaa gttatgtcga cggtgtggaa agtccccagg
      301 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
      361 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
      421 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
      481 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
      541 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
      601 attcgccacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
      661 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
      721 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
      781 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
      841 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
      901 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
      961 cttcgtggcc gaggagcagg actgactgca gcgactgtgc cttctagttg ccagccatct
     1021 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     1081 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     1141 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     1201 gatgcggtgg gctctatggc tcgaggaggg cctatttccc atgattcctt catatttgca
     1261 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
     1321 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     1381 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     1441 ggctttatat atcttgtgga aaggacgaaa caccgataac ttcgtatagc atacattata
     1501 cgaagttatg gtaccgatgc agctagcccg cggatctgcc ggtctcccta tagtgagtcg
     1561 tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc gcggggagag
     1621 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     1681 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     1741 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     1801 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     1861 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     1921 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     1981 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     2041 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2101 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2161 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2221 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     2281 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     2341 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     2401 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     2461 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     2521 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     2581 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     2641 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     2701 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     2761 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     2821 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     2881 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     2941 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     3001 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     3061 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     3121 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     3181 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     3241 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     3301 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     3361 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     3421 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     3481 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     3541 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     3601 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     3661 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     3721 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     3781 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg gacatattgt
     3841 cgttagaacg cggctacaat taatacataa ccttatgtat catacacata cg
//
