LOCUS Exported 3934 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with 3xFLAG. ACCESSION . VERSION . KEYWORDS pMaCTag-Z34 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3934) 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 3934) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3934 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..129 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 139..260 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(176..195) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 230..249 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 267..300 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 307..623 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 474..609 /label=SV40 ori /note="SV40 origin of replication" primer_bind 536..555 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 630..639 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 636..1010 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 1020..1244 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1251..1491 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1251..1271 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1422..1441 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1501..1534 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1571..1590) /label=T7 /note="T7 promoter, forward primer" promoter complement(1572..1590) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1677..1694) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1848..2436) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1928..1947) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2607..3467) /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 3230..3249 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3468..3572) /gene="bla" /label=AmpR promoter primer_bind 3640..3658 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3696..3718) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3818..3837 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 3918..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 3918..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccgactaca aagaccatga cggtgattat aaagatcatg atatcgatta caaggatgac 121 gatgacaagt agactagtaa cttgtttatt gcagcttata atggttacaa ataaagcaat 181 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 241 aaactcatca atgtatctta gtcgatataa cttcgtatag catacattat acgaagttat 301 gtcgacggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 361 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 421 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 481 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 541 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt 601 ttggaggcct aggcttttgc aaagaattcg ccaccatggc caagttgacc agtgccgttc 661 cggtgctcac cgcgcgcgac gtcgccggag cggtcgagtt ctggaccgac cggctcgggt 721 tctcccggga cttcgtggag gacgacttcg ccggtgtggt ccgggacgac gtgaccctgt 781 tcatcagcgc ggtccaggac caggtggtgc cggacaacac cctggcctgg gtgtgggtgc 841 gcggcctgga cgagctgtac gccgagtggt cggaggtcgt gtccacgaac ttccgggacg 901 cctccgggcc ggccatgacc gagatcggcg agcagccgtg ggggcgggag ttcgccctgc 961 gcgacccggc cggcaactgc gtgcacttcg tggccgagga gcaggactga ctgcagcgac 1021 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 1081 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 1141 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 1201 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggctcgag gagggcctat 1261 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa 1321 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat 1381 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg 1441 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg 1501 ataacttcgt atagcataca ttatacgaag ttatggtacc gatgcagcta gcccgcggat 1561 ctgccggtct ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa 1621 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 1681 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 1741 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 1801 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 1861 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 1921 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 1981 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 2041 caatgctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 2101 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 2161 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 2221 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 2281 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 2341 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 2401 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 2461 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 2521 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 2581 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 2641 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 2701 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 2761 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 2821 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 2881 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 2941 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 3001 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 3061 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 3121 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 3181 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 3241 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 3301 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 3361 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 3421 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 3481 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 3541 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 3601 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 3661 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag 3721 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca 3781 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg 3841 agagtgcacc atatggacat attgtcgtta gaacgcggct acaattaata cataacctta 3901 tgtatcatac acatacgatt taggtgacac tata //