LOCUS       sequence-228119-        4564 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Zeocin 
            selection) with TagBFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4564)
  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 4564)
  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-Z02
FEATURES             Location/Qualifiers
     source          1..4564
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             64..762
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    769..890
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(806..825)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     860..879
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(897..930)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        937..1253
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1104..1239
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      1260..1269
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1266..1640
                     /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    1650..1874
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        1881..2129
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1881..1901
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2052..2071
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2131..2164)
                     /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(2201..2220)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2202..2220)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2307..2324)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2478..3066)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2558..2577)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3237..4028,4029..4097))
                     /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     3860..3879
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4098..4202)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4270..4288
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4326..4348)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4448..4467
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4548..4564,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4548..4564,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac
      121 aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc
      181 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact
      241 agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc
      301 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc
      361 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag
      421 atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg
      481 gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg
      541 gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc
      601 aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa
      661 agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga
      721 tactgcgacc tccctagcaa actggggcac aagcttaatt gaactagtaa cttgtttatt
      781 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
      841 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta gtcgatataa
      901 cttcgtatag catacattat acgaagttat gtcgacggtg tggaaagtcc ccaggctccc
      961 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     1021 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     1081 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     1141 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     1201 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagaattcg
     1261 ccaccatggc caagttgacc agtgccgttc cggtgctcac cgcgcgcgac gtcgccggag
     1321 cggtcgagtt ctggaccgac cggctcgggt tctcccggga cttcgtggag gacgacttcg
     1381 ccggtgtggt ccgggacgac gtgaccctgt tcatcagcgc ggtccaggac caggtggtgc
     1441 cggacaacac cctggcctgg gtgtgggtgc gcggcctgga cgagctgtac gccgagtggt
     1501 cggaggtcgt gtccacgaac ttccgggacg cctccgggcc ggccatgacc gagatcggcg
     1561 agcagccgtg ggggcgggag ttcgccctgc gcgacccggc cggcaactgc gtgcacttcg
     1621 tggccgagga gcaggactga ctgcagcgac tgtgccttct agttgccagc catctgttgt
     1681 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1741 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1801 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     1861 ggtgggctct atggctcgag gagggcctat ttcccatgat tccttcatat ttgcatatac
     1921 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     1981 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2041 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2101 tatatatctt gtggaaagga cgaaacaccg ataacttcgt atagcataca ttatacgaag
     2161 ttatggtacc gatgcagcta gcccgcggat ctgccggtct ccctatagtg agtcgtatta
     2221 atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2281 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2341 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2401 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2461 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2521 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2581 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2641 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
     2701 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2761 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2821 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2881 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     2941 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3001 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3061 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3121 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3181 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3241 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3301 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3361 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3421 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3481 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3541 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3601 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3661 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3721 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3781 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3841 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3901 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3961 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4021 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4081 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4141 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4201 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     4261 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     4321 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     4381 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     4441 aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta
     4501 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
     4561 tata
//
