LOCUS       sequence-232486-        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"
     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..779
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     polyA_signal    786..907
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(823..842)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     877..896
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(914..947)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        954..1202
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     954..974
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1125..1144
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1204..1237)
                     /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(1274..1293)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1275..1293)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1380..1397)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1551..2139)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1631..1650)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2310..3101,3102..3170))
                     /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     2933..2952
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3171..3275)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3343..3361
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3399..3421)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3521..3540
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc agcgagctga ttaaggagaa catgcacatg aagctgtaca
      121 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct
      181 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct
      241 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg
      301 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca
      361 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc
      421 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga
      481 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg
      541 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca
      601 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg
      661 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg
      721 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattgaa
      781 ctagtaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
      841 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
      901 tatcttagtc gatataactt cgtatagcat acattatacg aagttatgtc gaggagggcc
      961 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
     1021 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     1081 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     1141 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     1201 ccgataactt cgtatagcat acattatacg aagttatggt accgatgcag ctagcccgcg
     1261 gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat
     1321 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     1381 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     1441 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     1501 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     1561 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     1621 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     1681 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     1741 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     1801 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     1861 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     1921 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     1981 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     2041 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     2101 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     2161 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     2221 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     2281 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     2341 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     2401 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     2461 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     2521 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     2581 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     2641 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     2701 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     2761 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     2821 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     2881 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     2941 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3001 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     3061 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3121 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3181 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3241 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3301 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     3361 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     3421 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     3481 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
     3541 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc
     3601 ttatgtatca tacacatacg
//
