LOCUS       sequence-228001-        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"
     CDS             64..717
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="VSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVT
                     MAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSAS
                     VSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYL
                     KLEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     polyA_signal    724..845
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(761..780)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     815..834
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(852..885)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        892..1140
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     892..912
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1063..1082
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1142..1175)
                     /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(1212..1231)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1213..1231)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1318..1335)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1489..2077)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1569..1588)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2248..3039,3040..3108))
                     /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     2871..2890
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3109..3213)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3281..3299
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3337..3359)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3459..3478
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(3559..3575,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(3559..3575,1)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga
       61 tccgtgagtg tgattaaacc agagatgaag atgaggtact acatggacgg ctccgtcaat
      121 gggcatgagt tcacaattga aggtgaaggc acaggcagac cttacgaggg acatcaagag
      181 atgacactac gcgtcacaat ggccgaaggc gggccaatgc ctttcgcgtt tgacttagtg
      241 tcacacgtgt tctgttacgg ccacagagtt tttactaaat atccagaaga gataccagac
      301 tatttcaaac aagcatttcc tgaaggcctg tcatgggaaa ggtcgttgga gttcgaagat
      361 ggtgggtccg cttcagtcag tgcgcatata agccttagag gaaacacctt ctaccacaaa
      421 tccaaattta ctggggttaa ctttcctgcc gatggtccta tcatgcaaaa ccaaagtgtt
      481 gattgggagc catcaaccga gaaaattact gccagcgacg gagttctgaa gggtgatgtt
      541 acgatgtacc taaaacttga aggaggcggc aatcacaaat gccaattcaa gactacttac
      601 aaggcggcaa aagaaattct tgaaatgcca ggagatcatt acatcggtca tcgcctcgtc
      661 aggaaaaccg aaggcaacat tactgagcaa gtagaagatg cagtagctca ttcctgaact
      721 agtaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
      781 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
      841 tcttagtcga tataacttcg tatagcatac attatacgaa gttatgtcga ggagggccta
      901 tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga gataattgga
      961 attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa
     1021 tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc
     1081 gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc
     1141 gataacttcg tatagcatac attatacgaa gttatggtac cgatgcagct agcccgcgga
     1201 tctgccggtc tccctatagt gagtcgtatt aatttcgata agccaggtta acctgcatta
     1261 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     1321 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     1381 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     1441 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     1501 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     1561 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     1621 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     1681 tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     1741 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     1801 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     1861 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     1921 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     1981 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2041 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     2101 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     2161 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     2221 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     2281 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     2341 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     2401 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     2461 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     2521 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     2581 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     2641 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     2701 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     2761 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     2821 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     2881 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     2941 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3001 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3061 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     3121 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     3181 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     3241 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     3301 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3361 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3421 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     3481 gagagtgcac catatggaca tattgtcgtt agaacgcggc tacaattaat acataacctt
     3541 atgtatcata cacatacgat ttaggtgaca ctata
//
