LOCUS Exported 3629 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (without selection) with mCherry. ACCESSION . VERSION . KEYWORDS pMaCTag-13 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3629) 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 3629) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3629 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..771 /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(206..224) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 474..493 /label=mCherry-F /note="mCherry, forward primer" polyA_signal 778..899 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(815..834) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 869..888 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 906..939 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 946..1186 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 946..966 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1117..1136 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1196..1229 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1266..1285) /label=T7 /note="T7 promoter, forward primer" promoter complement(1267..1285) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1372..1389) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1543..2131) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1623..1642) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2302..3162) /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 2925..2944 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3163..3267) /gene="bla" /label=AmpR promoter primer_bind 3335..3353 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3391..3413) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3513..3532 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 3613..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 3613..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccgtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 121 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 181 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 241 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 301 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 361 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 421 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 481 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 541 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 601 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 661 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 721 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagtg aactagtaac 781 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 841 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag 901 tcgatataac ttcgtatagc atacattata cgaagttatg tcgaggaggg cctatttccc 961 atgattcctt catatttgca tatacgatac aaggctgtta gagagataat tggaattaat 1021 ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt 1081 gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact 1141 tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgataac 1201 ttcgtatagc atacattata cgaagttatg gtaccgatgc agctagcccg cggatctgcc 1261 ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat 1321 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 1381 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 1441 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 1501 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 1561 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 1621 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 1681 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg 1741 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 1801 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 1861 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 1921 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 1981 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 2041 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 2101 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 2161 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 2221 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 2281 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 2341 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 2401 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 2461 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 2521 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 2581 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 2641 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 2701 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 2761 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 2821 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 2881 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 2941 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 3001 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 3061 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 3121 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 3181 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 3241 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta 3301 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 3361 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 3421 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 3481 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt 3541 gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat 3601 catacacata cgatttaggt gacactata //