LOCUS       sequence-232610-        4789 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with TagBFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4789)
  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 4789)
  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-P02
FEATURES             Location/Qualifiers
     source          1..4789
                     /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..1270
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1121..1256
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1892..2116
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2123..2371
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2123..2143
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2294..2313
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2373..2406)
                     /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(2443..2462)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2444..2462)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2549..2566)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2720..3308)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2800..2819)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3479..4270,4271..4339))
                     /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     4102..4121
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4340..4444)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4512..4530
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4568..4590)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4690..4709
                     /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 gacggtgtgg
      961 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     1021 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     1081 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     1141 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     1201 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     1261 cttttgcaaa gaattcgcca ccatgactga gtacaagcct actgtgcgtt tagccaccag
     1321 ggatgatgtg ccacgagccg tgaggacact agcagccgcc tttgctgact acccagctac
     1381 aaggcacacc gtggaccctg acagacacat tgaaagagtc acagaacttc aggagctgtt
     1441 tttaactagg gtgggcctgg atattggcaa agtctgggtg gccgatgatg gagcagcagt
     1501 tgcagtgtgg accaccccag agtctgttga agctggagct gtctttgcag aaattggacc
     1561 aagaatggcc gagttatctg gcagcaggct ggctgctcaa cagcagatgg aaggattatt
     1621 agctccccac agacccaagg agcctgcctg gttcctggca acagttggag tttctccaga
     1681 ccatcagggc aagggcttag gatctgctgt ggtgctgcct ggtgtggaag cagctgagag
     1741 agctggggtg ccagcatttc tggagacatc agctcctaga aacctgccct tctatgaacg
     1801 gctgggcttc acagtgactg ctgatgtgga ggtgcctgag ggccccagaa cctggtgcat
     1861 gacaagaaaa ccaggagcct gactgcagcg actgtgcctt ctagttgcca gccatctgtt
     1921 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     1981 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     2041 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2101 gcggtgggct ctatggctcg aggagggcct atttcccatg attccttcat atttgcatat
     2161 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
     2221 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     2281 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     2341 tttatatatc ttgtggaaag gacgaaacac cgataacttc gtatagcata cattatacga
     2401 agttatggta ccgatgcagc tagcccgcgg atctgccggt ctccctatag tgagtcgtat
     2461 taatttcgat aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     2521 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     2581 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     2641 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     2701 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     2761 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     2821 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     2881 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     2941 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     3001 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     3061 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     3121 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     3181 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     3241 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     3301 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     3361 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     3421 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     3481 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     3541 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     3601 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     3661 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     3721 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     3781 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     3841 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     3901 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     3961 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     4021 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     4081 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     4141 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     4201 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     4261 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     4321 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     4381 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     4441 cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat
     4501 taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg
     4561 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     4621 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc
     4681 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt
     4741 tagaacgcgg ctacaattaa tacataacct tatgtatcat acacatacg
//
