LOCUS Exported 3467 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (without selection) with TAP. ACCESSION . VERSION . KEYWORDS pMaCTag-24 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 May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3467 /organism="synthetic DNA construct" /mol_type="other DNA" 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..158 /codon_start=1 /product="calmodulin-binding peptide" /label=CBP /note="derived from skeletal muscle myosin light chain kinase; binds calmodulin with nanomolar affinity in the presence of calcium" /translation="KRRWKKNFIAVSAANRFKKISSSGAL" CDS 186..206 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQG" CDS 246..419 /codon_start=1 /product="IgG-binding unit of Staphylococcus aureus protein A" /label=ProtA /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL AEAKKLNDAQAPK" CDS 423..593 /codon_start=1 /product="IgG-binding unit of Staphylococcus aureus protein A" /label=ProtA /translation="DNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLA EAKKLNGAQAPK" 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 761..794 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 801..1041 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" 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 1051..1084 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." 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(2157..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 aagagaagat ggaaaaagaa tttcatagcc gtctcagcag 121 ccaaccgctt taagaaaatc tcatcctccg gggcacttga ttatgatatt ccaactactg 181 ctagcgagaa tttgtatttt cagggtgagc tcaaaaccgc ggctcttgcg caacacgatg 241 aagccgtgga caacaaattc aacaaagaac aacaaaacgc gttctatgag atcttacatt 301 tacctaactt aaacgaagaa caacgaaacg ccttcatcca aagtttaaaa gatgacccaa 361 gccaaagcgc taacctttta gcagaagcta aaaagctaaa tgatgctcag gcgccgaaag 421 tagacaacaa attcaacaaa gaacaacaaa acgcgttcta tgagatctta catttaccta 481 acttaaacga agaacaacga aacgccttca tccaaagttt aaaagatgac ccaagccaaa 541 gcgctaacct tttagcagaa gctaaaaagc taaatggtgc tcaggcgccg aaagtagacg 601 cgaattccgc ggggaagtca acctgaacta 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 //