LOCUS       sequence-232599-        3467 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template for C-terminal PCR tagging of mammalian genes (without 
            selection) with SNAP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3467)
  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 3467)
  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-22
FEATURES             Location/Qualifiers
     source          1..3467
                     /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..623
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="optimized for mammalian expression"
                     /translation="DKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVEV
                     PAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKV
                     VKFGEVISYQQLAALAGNPAATAAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLAV
                     KEWLLAHEGHRLGKPGLG"
     polyA_signal    633..754
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(670..689)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     724..743
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(761..794)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        801..1049
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     801..821
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     972..991
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     protein_bind    complement(1051..1084)
                     /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(1121..1140)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1122..1140)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1227..1244)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1398..1986)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1478..1497)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2157..2948,2949..3017))
                     /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     2780..2799
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3018..3122)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3190..3208
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3246..3268)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3368..3387
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag
       61 gtagtggcgg aggcggatcc gacaaagact gcgaaatgaa gcgcaccacc ctggatagcc
      121 ctctgggcaa gctggaactg tctgggtgcg aacagggcct gcacgagatc aagctgctgg
      181 gcaaaggaac atctgccgcc gacgccgtgg aagtgcctgc cccagccgcc gtgctgggcg
      241 gaccagagcc actgatgcag gccaccgcct ggctcaacgc ctactttcac cagcctgagg
      301 ccatcgagga gttccctgtg ccagccctgc accacccagt gttccagcag gagagcttta
      361 cccgccaggt gctgtggaaa ctgctgaaag tggtgaagtt cggagaggtc atcagctacc
      421 agcagctggc cgccctggcc ggcaatcccg ccgccaccgc cgccgtgaaa accgccctga
      481 gcggaaatcc cgtgcccatt ctgatcccct gccaccgggt ggtgtctagc tctggcgccg
      541 tggggggcta cgagggcggg ctcgccgtga aagagtggct gctggcccac gagggccaca
      601 gactgggcaa gcctgggctg ggttgaacta gtaacttgtt tattgcagct tataatggtt
      661 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
      721 gttgtggttt gtccaaactc atcaatgtat cttagtcgat ataacttcgt atagcataca
      781 ttatacgaag ttatgtcgag gagggcctat ttcccatgat tccttcatat ttgcatatac
      841 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
      901 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
      961 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
     1021 tatatatctt gtggaaagga cgaaacaccg ataacttcgt atagcataca ttatacgaag
     1081 ttatggtacc gatgcagcta gcccgcggat ctgccggtct ccctatagtg agtcgtatta
     1141 atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1201 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1261 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1321 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1381 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1441 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1501 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1561 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1621 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     1681 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     1741 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     1801 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     1861 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     1921 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     1981 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2041 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2101 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2161 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2221 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2281 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     2341 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2401 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2461 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     2521 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     2581 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     2641 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     2701 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     2761 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     2821 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     2881 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     2941 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3001 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3061 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3121 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     3181 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt
     3241 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     3301 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt
     3361 aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta
     3421 gaacgcggct acaattaata cataacctta tgtatcatac acatacg
//
