LOCUS       sequence-227990-        3620 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with TagBFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3620)
  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 3620)
  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-02
FEATURES             Location/Qualifiers
     source          1..3620
                     /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..1185
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     937..957
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1108..1127
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1187..1220)
                     /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(1257..1276)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1258..1276)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1363..1380)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1534..2122)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1614..1633)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2293..3084,3085..3153))
                     /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     2916..2935
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3154..3258)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3326..3344
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3382..3404)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3504..3523
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3604..3620,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3604..3620,1)
                     /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 gtcgaggagg gcctatttcc catgattcct
      961 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta
     1021 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt
     1081 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat
     1141 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgataa cttcgtatag
     1201 catacattat acgaagttat ggtaccgatg cagctagccc gcggatctgc cggtctccct
     1261 atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg
     1321 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     1381 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     1441 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     1501 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     1561 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     1621 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     1681 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg
     1741 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     1801 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     1861 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     1921 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     1981 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     2041 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     2101 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     2161 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     2221 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     2281 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     2341 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     2401 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     2461 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     2521 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     2581 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     2641 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     2701 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     2761 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     2821 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     2881 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     2941 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     3001 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     3061 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     3121 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     3181 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3241 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     3301 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     3361 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     3421 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     3481 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     3541 ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat
     3601 acgatttagg tgacactata
//
