LOCUS       sequence-228039-        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"
     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"
     polyA_signal    1875..2099
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2106..2354
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2106..2126
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2277..2296
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2356..2389)
                     /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(2426..2445)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2427..2445)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2532..2549)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2703..3291)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2783..2802)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3462..4253,4254..4322))
                     /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     4085..4104
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4323..4427)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4495..4513
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4551..4573)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4673..4692
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4773..4789,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4773..4789,1)
                     /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 ccaccatgac tgagtacaag cctactgtgc gtttagccac cagggatgat gtgccacgag
     1321 ccgtgaggac actagcagcc gcctttgctg actacccagc tacaaggcac accgtggacc
     1381 ctgacagaca cattgaaaga gtcacagaac ttcaggagct gtttttaact agggtgggcc
     1441 tggatattgg caaagtctgg gtggccgatg atggagcagc agttgcagtg tggaccaccc
     1501 cagagtctgt tgaagctgga gctgtctttg cagaaattgg accaagaatg gccgagttat
     1561 ctggcagcag gctggctgct caacagcaga tggaaggatt attagctccc cacagaccca
     1621 aggagcctgc ctggttcctg gcaacagttg gagtttctcc agaccatcag ggcaagggct
     1681 taggatctgc tgtggtgctg cctggtgtgg aagcagctga gagagctggg gtgccagcat
     1741 ttctggagac atcagctcct agaaacctgc ccttctatga acggctgggc ttcacagtga
     1801 ctgctgatgt ggaggtgcct gagggcccca gaacctggtg catgacaaga aaaccaggag
     1861 cctgactgca gcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     1921 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     1981 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     2041 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     2101 tcgaggaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     2161 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     2221 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     2281 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     2341 aaggacgaaa caccgataac ttcgtatagc atacattata cgaagttatg gtaccgatgc
     2401 agctagcccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag
     2461 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2521 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2581 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2641 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2701 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2761 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2821 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2881 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2941 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     3001 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     3061 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3121 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     3181 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3241 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3301 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3361 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3421 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3481 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3541 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3601 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3661 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3721 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3781 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3841 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3901 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3961 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     4021 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     4081 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4141 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4201 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4261 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4321 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4381 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4441 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     4501 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     4561 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     4621 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     4681 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat
     4741 taatacataa ccttatgtat catacacata cgatttaggt gacactata
//
