LOCUS Exported 4117 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with Strep-II. ACCESSION . VERSION . KEYWORDS pMaCTag-P29 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4117) 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 4117) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4117 /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..104 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" polyA_signal 114..235 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(151..170) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 205..224 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 242..275 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 282..598 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 449..584 /label=SV40 ori /note="SV40 origin of replication" primer_bind 511..530 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 1220..1444 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1451..1691 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1451..1471 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1622..1641 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1701..1734 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1771..1790) /label=T7 /note="T7 promoter, forward primer" promoter complement(1772..1790) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1877..1894) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2048..2636) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2128..2147) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2807..3667) /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 3430..3449 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3668..3772) /gene="bla" /label=AmpR promoter primer_bind 3840..3858 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3896..3918) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4018..4037 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc tggagccacc cccagttcga gaagtgaact agtaacttgt 121 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 181 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttagtcga 241 tataacttcg tatagcatac attatacgaa gttatgtcga cggtgtggaa agtccccagg 301 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg 361 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc 421 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca 481 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc 541 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga 601 attcgccacc atgactgagt acaagcctac tgtgcgttta gccaccaggg atgatgtgcc 661 acgagccgtg aggacactag cagccgcctt tgctgactac ccagctacaa ggcacaccgt 721 ggaccctgac agacacattg aaagagtcac agaacttcag gagctgtttt taactagggt 781 gggcctggat attggcaaag tctgggtggc cgatgatgga gcagcagttg cagtgtggac 841 caccccagag tctgttgaag ctggagctgt ctttgcagaa attggaccaa gaatggccga 901 gttatctggc agcaggctgg ctgctcaaca gcagatggaa ggattattag ctccccacag 961 acccaaggag cctgcctggt tcctggcaac agttggagtt tctccagacc atcagggcaa 1021 gggcttagga tctgctgtgg tgctgcctgg tgtggaagca gctgagagag ctggggtgcc 1081 agcatttctg gagacatcag ctcctagaaa cctgcccttc tatgaacggc tgggcttcac 1141 agtgactgct gatgtggagg tgcctgaggg ccccagaacc tggtgcatga caagaaaacc 1201 aggagcctga ctgcagcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc 1261 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag 1321 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag 1381 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct 1441 atggctcgag gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc 1501 tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac 1561 gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat 1621 ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt 1681 gtggaaagga cgaaacaccg ataacttcgt atagcataca ttatacgaag ttatggtacc 1741 gatgcagcta gcccgcggat ctgccggtct ccctatagtg agtcgtatta atttcgataa 1801 gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 1861 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 1921 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 1981 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 2041 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 2101 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 2161 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 2221 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 2281 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 2341 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 2401 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 2461 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 2521 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 2581 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 2641 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 2701 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 2761 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 2821 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 2881 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 2941 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 3001 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 3061 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 3121 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 3181 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 3241 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 3301 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 3361 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 3421 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 3481 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 3541 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 3601 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 3661 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 3721 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 3781 cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 3841 ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct 3901 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac 3961 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg 4021 catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta gaacgcggct 4081 acaattaata cataacctta tgtatcatac acatacg //