LOCUS       sequence-232506-        3575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with mKOk.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3575)
  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 3575)
  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-10
FEATURES             Location/Qualifiers
     source          1..3575
                     /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..734
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="VSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVT
                     MAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSAS
                     VSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL
                     KLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    741..862
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(778..797)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     832..851
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(869..902)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        909..1157
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     909..929
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1080..1099
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1159..1192)
                     /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(1229..1248)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1230..1248)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1335..1352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1506..2094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1586..1605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2265..3056,3057..3125))
                     /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     2888..2907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3126..3230)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3298..3316
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3354..3376)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3476..3495
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gtgagtgtga ttaaaccaga gatgaagatg aggtactaca
      121 tggacggctc cgtcaatggg catgagttca caattgaagg tgaaggcaca ggcagacctt
      181 acgagggaca tcaagagatg acactacgcg tcacaatggc cgaaggcggg ccaatgcctt
      241 tcgcgtttga cttagtgtca cacgtgttct gttacggcca cagagttttt actaaatatc
      301 cagaagagat accagactat ttcaaacaag catttcctga aggcctgtca tgggaaaggt
      361 cgttggagtt cgaagatggt gggtccgctt cagtcagtgc gcatataagc cttagaggaa
      421 acaccttcta ccacaaatcc aaatttactg gggttaactt tcctgccgat ggtcctatca
      481 tgcaaaacca aagtgttgat tgggagccat caaccgagaa aattactgcc agcgacggag
      541 ttctgaaggg tgatgttacg atgtacctaa aacttgaagg aggcggcaat cacaaatgcc
      601 aattcaagac tacttacaag gcggcaaaag aaattcttga aatgccagga gatcattaca
      661 tcggtcatcg cctcgtcagg aaaaccgaag gcaacattac tgagcaagta gaagatgcag
      721 tagctcattc ctgaactagt aacttgttta ttgcagctta taatggttac aaataaagca
      781 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
      841 ccaaactcat caatgtatct tagtcgatat aacttcgtat agcatacatt atacgaagtt
      901 atgtcgagga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg
      961 ttagagagat aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt
     1021 gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg
     1081 actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt
     1141 ggaaaggacg aaacaccgat aacttcgtat agcatacatt atacgaagtt atggtaccga
     1201 tgcagctagc ccgcggatct gccggtctcc ctatagtgag tcgtattaat ttcgataagc
     1261 caggttaacc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1321 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1381 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1441 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1501 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1561 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1621 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1681 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1741 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1801 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1861 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1921 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     1981 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2041 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2101 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2161 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2221 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2281 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2341 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2401 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2461 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2521 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2581 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2641 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2701 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2761 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     2821 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     2881 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     2941 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3001 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3061 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3121 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3181 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3241 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     3301 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     3361 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     3421 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     3481 tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga acgcggctac
     3541 aattaataca taaccttatg tatcatacac atacg
//
