LOCUS Exported 4561 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with mScarlet. ACCESSION . VERSION . KEYWORDS pMaCTag-Z12 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4561) 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 4561) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4561 /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..773 /codon_start=1 /product="bright monomeric red fluorescent protein evolved from a synthetic template (Bindels et al., 2016)" /label=mScarlet /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGGA VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS TGGMDELYK" primer_bind complement(211..229) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 479..498 /label=mCherry-F /note="mCherry, forward primer" polyA_signal 783..904 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(820..839) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 874..893 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 911..944 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 951..1267 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1118..1253 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1180..1199 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 1274..1283 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1280..1654 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 1664..1888 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1895..2135 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1895..1915 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2066..2085 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2145..2178 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2215..2234) /label=T7 /note="T7 promoter, forward primer" promoter complement(2216..2234) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2321..2338) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2492..3080) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2572..2591) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3251..4111) /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 3874..3893 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4112..4216) /gene="bla" /label=AmpR promoter primer_bind 4284..4302 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4340..4362) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4462..4481 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggcagt gatcaaggag ttcatgcggt 121 tcaaggtgca catggagggc tccatgaacg gccacgagtt cgagatcgag ggcgagggcg 181 agggccgccc ctacgagggc acccagaccg ccaagctgaa ggtgaccaag ggtggccccc 241 tgcccttctc ctgggacatc ctgtcccctc agttcatgta cggctccagg gccttcacca 301 agcaccccgc cgacatcccc gactactata agcagtcctt ccccgagggc ttcaagtggg 361 agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt gacccaggac acctccctgg 421 aggacggcac cctgatctac aaggtgaagc tccgcggcac caacttccct cctgacggcc 481 ccgtaatgca gaagaagaca atgggctggg aagcgtccac cgagcggttg taccccgagg 541 acggcgtgct gaagggcgac attaagatgg ccctgcgcct gaaggacggc ggccgctacc 601 tggcggactt caagaccacc tacaaggcca agaagcccgt gcagatgccc ggcgcctaca 661 acgtcgaccg caagttggac atcacctccc acaacgagga ctacaccgtg gtggaacagt 721 acgaacgctc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagtgaacta 781 gtaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 841 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 901 cttagtcgat ataacttcgt atagcataca ttatacgaag ttatgtcgac ggtgtggaaa 961 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 1021 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 1081 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 1141 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 1201 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 1261 ttgcaaagaa ttcgccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg 1321 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt 1381 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca 1441 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct 1501 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat 1561 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa 1621 ctgcgtgcac ttcgtggccg aggagcagga ctgactgcag cgactgtgcc ttctagttgc 1681 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 1741 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 1801 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 1861 catgctgggg atgcggtggg ctctatggct cgaggagggc ctatttccca tgattccttc 1921 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa 1981 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc 2041 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt 2101 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgataact tcgtatagca 2161 tacattatac gaagttatgg taccgatgca gctagcccgc ggatctgccg gtctccctat 2221 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg 2281 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 2341 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 2401 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 2461 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 2521 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 2581 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 2641 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 2701 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 2761 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 2821 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 2881 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 2941 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 3001 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 3061 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 3121 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 3181 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 3241 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 3301 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 3361 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 3421 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 3481 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 3541 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 3601 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 3661 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 3721 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 3781 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 3841 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 3901 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 3961 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 4021 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 4081 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 4141 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 4201 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 4261 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt 4321 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 4381 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 4441 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg 4501 acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac 4561 g //