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" 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..779 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD LPSKLGHKLN" polyA_signal 786..907 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(823..842) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 877..896 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 914..947 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 954..1194 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 954..974 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1125..1144 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1204..1237 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1274..1293) /label=T7 /note="T7 promoter, forward primer" promoter complement(1275..1293) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1380..1397) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1551..2139) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1631..1650) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2310..3170) /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 2933..2952 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3171..3275) /gene="bla" /label=AmpR promoter primer_bind 3343..3361 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3399..3421) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3521..3540 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc agcgagctga ttaaggagaa catgcacatg aagctgtaca 121 tggagggcac cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct 181 acgagggcac ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgcct 241 tcgacatcct ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacccagg 301 gcatccccga cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca 361 catacgaaga cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc 421 tcatctacaa cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga 481 agaaaacact cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg 541 aaggcagaaa cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca 601 agaccacata tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg 661 tggactacag actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg 721 aggtggcagt ggccagatac tgcgacctcc ctagcaaact ggggcacaag cttaattgaa 781 ctagtaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 841 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 901 tatcttagtc gatataactt cgtatagcat acattatacg aagttatgtc gaggagggcc 961 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg 1021 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat 1081 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta 1141 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca 1201 ccgataactt cgtatagcat acattatacg aagttatggt accgatgcag ctagcccgcg 1261 gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat 1321 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 1381 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 1441 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 1501 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 1561 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 1621 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 1681 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 1741 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1801 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1861 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1921 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1981 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 2041 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 2101 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 2161 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 2221 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 2281 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 2341 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 2401 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 2461 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 2521 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 2581 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 2641 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 2701 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 2761 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2821 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 2881 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2941 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 3001 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 3061 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 3121 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 3181 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 3241 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 3301 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 3361 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg 3421 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg 3481 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta 3541 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc 3601 ttatgtatca tacacatacg //