LOCUS       sequence-228047-        4744 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (Puromycin 
            selection) with mKOk.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4744)
  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 4744)
  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-P10
FEATURES             Location/Qualifiers
     source          1..4744
                     /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..1208
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1059..1194
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    1830..2054
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        2061..2309
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2061..2081
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2232..2251
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(2311..2344)
                     /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(2381..2400)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2382..2400)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2487..2504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2658..3246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2738..2757)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3417..4208,4209..4277))
                     /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     4040..4059
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4278..4382)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4450..4468
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4506..4528)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4628..4647
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        join(4728..4744,1..2)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     join(4728..4744,1)
                     /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 cggtgtggaa
      901 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
      961 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     1021 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     1081 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     1141 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     1201 tttgcaaaga attcgccacc atgactgagt acaagcctac tgtgcgttta gccaccaggg
     1261 atgatgtgcc acgagccgtg aggacactag cagccgcctt tgctgactac ccagctacaa
     1321 ggcacaccgt ggaccctgac agacacattg aaagagtcac agaacttcag gagctgtttt
     1381 taactagggt gggcctggat attggcaaag tctgggtggc cgatgatgga gcagcagttg
     1441 cagtgtggac caccccagag tctgttgaag ctggagctgt ctttgcagaa attggaccaa
     1501 gaatggccga gttatctggc agcaggctgg ctgctcaaca gcagatggaa ggattattag
     1561 ctccccacag acccaaggag cctgcctggt tcctggcaac agttggagtt tctccagacc
     1621 atcagggcaa gggcttagga tctgctgtgg tgctgcctgg tgtggaagca gctgagagag
     1681 ctggggtgcc agcatttctg gagacatcag ctcctagaaa cctgcccttc tatgaacggc
     1741 tgggcttcac agtgactgct gatgtggagg tgcctgaggg ccccagaacc tggtgcatga
     1801 caagaaaacc aggagcctga ctgcagcgac tgtgccttct agttgccagc catctgttgt
     1861 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     1921 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     1981 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2041 ggtgggctct atggctcgag gagggcctat ttcccatgat tccttcatat ttgcatatac
     2101 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
     2161 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
     2221 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     2281 tatatatctt gtggaaagga cgaaacaccg ataacttcgt atagcataca ttatacgaag
     2341 ttatggtacc gatgcagcta gcccgcggat ctgccggtct ccctatagtg agtcgtatta
     2401 atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2461 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     2521 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     2581 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     2641 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     2701 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     2761 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     2821 tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg
     2881 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     2941 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3001 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3061 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     3121 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3181 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3241 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3301 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3361 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3421 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3481 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     3541 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     3601 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     3661 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     3721 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     3781 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     3841 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     3901 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3961 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4021 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4081 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4141 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4201 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4261 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4321 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4381 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     4441 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     4501 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     4561 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     4621 aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta
     4681 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
     4741 tata
//
