LOCUS Exported 4807 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with mVenus. ACCESSION . VERSION . KEYWORDS pMaCTag-P09 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4807) 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 4807) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4807 /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..797 /codon_start=1 /product="Venus YFP with monomerizing A206K mutation (Nagai et al., 2002; Kremers et al., 2006)" /label=mVenus /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK LICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(123..144) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(384..403) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 731..752 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 804..925 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(841..860) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 895..914 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 932..965 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 972..1288 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1139..1274 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1201..1220 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 1910..2134 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 2141..2381 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2141..2161 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2312..2331 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2391..2424 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2461..2480) /label=T7 /note="T7 promoter, forward primer" promoter complement(2462..2480) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2567..2584) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2738..3326) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2818..2837) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3497..4357) /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 4120..4139 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4358..4462) /gene="bla" /label=AmpR promoter primer_bind 4530..4548 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4586..4608) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4708..4727 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 121 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 181 agggcgatgc cacctacggc aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc 241 ccgtgccctg gcccaccctc gtgaccaccc tgggctacgg cctgcagtgc ttcgcccgct 301 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 361 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 421 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 481 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcaccg 541 ccgacaagca gaagaacggc atcaaggcca acttcaagat ccgccacaac atcgaggacg 601 gcggcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 661 tgctgcccga caaccactac ctgagctacc agtccaagct gagcaaagac cccaacgaga 721 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 781 acgagctgta caagtgaact agtaacttgt ttattgcagc ttataatggt tacaaataaa 841 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 901 tgtccaaact catcaatgta tcttagtcga tataacttcg tatagcatac attatacgaa 961 gttatgtcga cggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 1021 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 1081 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 1141 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 1201 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg 1261 cttttttgga ggcctaggct tttgcaaaga attcgccacc atgactgagt acaagcctac 1321 tgtgcgttta gccaccaggg atgatgtgcc acgagccgtg aggacactag cagccgcctt 1381 tgctgactac ccagctacaa ggcacaccgt ggaccctgac agacacattg aaagagtcac 1441 agaacttcag gagctgtttt taactagggt gggcctggat attggcaaag tctgggtggc 1501 cgatgatgga gcagcagttg cagtgtggac caccccagag tctgttgaag ctggagctgt 1561 ctttgcagaa attggaccaa gaatggccga gttatctggc agcaggctgg ctgctcaaca 1621 gcagatggaa ggattattag ctccccacag acccaaggag cctgcctggt tcctggcaac 1681 agttggagtt tctccagacc atcagggcaa gggcttagga tctgctgtgg tgctgcctgg 1741 tgtggaagca gctgagagag ctggggtgcc agcatttctg gagacatcag ctcctagaaa 1801 cctgcccttc tatgaacggc tgggcttcac agtgactgct gatgtggagg tgcctgaggg 1861 ccccagaacc tggtgcatga caagaaaacc aggagcctga ctgcagcgac tgtgccttct 1921 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc 1981 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt 2041 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat 2101 agcaggcatg ctggggatgc ggtgggctct atggctcgag gagggcctat ttcccatgat 2161 tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 2221 tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 2281 gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 2341 gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg ataacttcgt 2401 atagcataca ttatacgaag ttatggtacc gatgcagcta gcccgcggat ctgccggtct 2461 ccctatagtg agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc 2521 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 2581 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 2641 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 2701 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 2761 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 2821 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 2881 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 2941 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 3001 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 3061 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 3121 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 3181 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 3241 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga 3301 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg 3361 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct 3421 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt 3481 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 3541 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 3601 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag 3661 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt 3721 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag 3781 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt 3841 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca 3901 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg 3961 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat 4021 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta 4081 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca 4141 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct 4201 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat 4261 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa 4321 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt 4381 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa 4441 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa 4501 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg 4561 cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag 4621 cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg 4681 gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc 4741 atatggacat attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac 4801 acatacg //