LOCUS Exported 4798 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with mCherry. ACCESSION . VERSION . KEYWORDS pMaCTag-P13 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4798) 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 4798) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4798 /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..788 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE GRHSTGGMDELYK" primer_bind complement(223..241) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 491..510 /label=mCherry-F /note="mCherry, forward primer" polyA_signal 795..916 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(832..851) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 886..905 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 923..956 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 963..1279 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1130..1265 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1192..1211 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 1901..2125 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 2132..2372 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2132..2152 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2303..2322 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2382..2415 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2452..2471) /label=T7 /note="T7 promoter, forward primer" promoter complement(2453..2471) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2558..2575) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2729..3317) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2809..2828) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3488..4348) /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 4111..4130 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4349..4453) /gene="bla" /label=AmpR promoter primer_bind 4521..4539 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4577..4599) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4699..4718 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagga taacatggcc atcatcaagg 121 agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg 181 agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca 241 agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca 301 aggcctacgt gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg 361 gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg 421 actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc 481 cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga 541 tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg 601 gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc 661 ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca 721 tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt 781 acaagtgaac tagtaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 841 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 901 tcatcaatgt atcttagtcg atataacttc gtatagcata cattatacga agttatgtcg 961 acggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 1021 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 1081 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 1141 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 1201 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg 1261 aggcctaggc ttttgcaaag aattcgccac catgactgag tacaagccta ctgtgcgttt 1321 agccaccagg gatgatgtgc cacgagccgt gaggacacta gcagccgcct ttgctgacta 1381 cccagctaca aggcacaccg tggaccctga cagacacatt gaaagagtca cagaacttca 1441 ggagctgttt ttaactaggg tgggcctgga tattggcaaa gtctgggtgg ccgatgatgg 1501 agcagcagtt gcagtgtgga ccaccccaga gtctgttgaa gctggagctg tctttgcaga 1561 aattggacca agaatggccg agttatctgg cagcaggctg gctgctcaac agcagatgga 1621 aggattatta gctccccaca gacccaagga gcctgcctgg ttcctggcaa cagttggagt 1681 ttctccagac catcagggca agggcttagg atctgctgtg gtgctgcctg gtgtggaagc 1741 agctgagaga gctggggtgc cagcatttct ggagacatca gctcctagaa acctgccctt 1801 ctatgaacgg ctgggcttca cagtgactgc tgatgtggag gtgcctgagg gccccagaac 1861 ctggtgcatg acaagaaaac caggagcctg actgcagcga ctgtgccttc tagttgccag 1921 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 1981 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 2041 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 2101 gctggggatg cggtgggctc tatggctcga ggagggccta tttcccatga ttccttcata 2161 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac 2221 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt 2281 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga 2341 tttcttggct ttatatatct tgtggaaagg acgaaacacc gataacttcg tatagcatac 2401 attatacgaa gttatggtac cgatgcagct agcccgcgga tctgccggtc tccctatagt 2461 gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc caacgcgcgg 2521 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 2581 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 2641 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 2701 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 2761 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 2821 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 2881 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2941 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 3001 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 3061 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 3121 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 3181 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 3241 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 3301 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 3361 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 3421 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 3481 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 3541 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 3601 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 3661 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 3721 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 3781 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 3841 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 3901 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 3961 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 4021 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 4081 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 4141 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 4201 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 4261 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 4321 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 4381 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 4441 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta 4501 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg 4561 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt 4621 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc 4681 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatggaca 4741 tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata cacatacg //