LOCUS Exported 3620 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (without selection) with TagBFP. ACCESSION . VERSION . KEYWORDS pMaCTag-02 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3620) 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 3620) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3620 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..762 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD LPSKLGHKLN" polyA_signal 769..890 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(806..825) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 860..879 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 897..930 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 937..1177 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 937..957 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1108..1127 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1187..1220 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1257..1276) /label=T7 /note="T7 promoter, forward primer" promoter complement(1258..1276) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1363..1380) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1534..2122) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1614..1633) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2293..3153) /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 2916..2935 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3154..3258) /gene="bla" /label=AmpR promoter primer_bind 3326..3344 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3382..3404) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3504..3523 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 3604..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 3604..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac 121 aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc 181 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact 241 agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc 301 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc 361 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag 421 atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg 481 gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg 541 gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc 601 aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa 661 agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga 721 tactgcgacc tccctagcaa actggggcac aagcttaatt gaactagtaa cttgtttatt 781 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 841 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta gtcgatataa 901 cttcgtatag catacattat acgaagttat gtcgaggagg gcctatttcc catgattcct 961 tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa tttgactgta 1021 aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct tgggtagttt 1081 gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac ttgaaagtat 1141 ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgataa cttcgtatag 1201 catacattat acgaagttat ggtaccgatg cagctagccc gcggatctgc cggtctccct 1261 atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa tcggccaacg 1321 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 1381 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 1441 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 1501 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 1561 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 1621 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 1681 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg 1741 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 1801 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 1861 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 1921 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 1981 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 2041 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 2101 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 2161 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 2221 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 2281 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 2341 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 2401 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 2461 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 2521 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 2581 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 2641 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 2701 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 2761 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 2821 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 2881 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 2941 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 3001 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 3061 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 3121 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 3181 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 3241 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat 3301 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg 3361 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg 3421 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg 3481 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat 3541 ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta tcatacacat 3601 acgatttagg tgacactata //