LOCUS Exported 4573 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with 10xSuntag. ACCESSION . VERSION . KEYWORDS pMaCTag-Z20 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4573) 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 4573) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4573 /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..137 /codon_start=1 /product="GCN4 peptide optimized to improve solubility while preserving binding to an scFv-GFP fusion protein (Tanenbaum et al., 2014)" /label=GCN4_v4 /translation="EELLSKNYHLENEVARLKK" CDS 153..209 /codon_start=1 /product="GCN4 peptide optimized to improve solubility while preserving binding to an scFv-GFP fusion protein (Tanenbaum et al., 2014)" /label=GCN4_v4 /translation="EELLSKNYHLENEVARLKK" CDS 225..281 /codon_start=1 /product="GCN4 peptide optimized to improve solubility while preserving binding to an scFv-GFP fusion protein (Tanenbaum et al., 2014)" /label=GCN4_v4 /translation="EELLSKNYHLENEVARLKK" CDS 297..353 /codon_start=1 /product="GCN4 peptide optimized to improve solubility while preserving binding to an scFv-GFP fusion protein (Tanenbaum et al., 2014)" /label=GCN4_v4 /translation="EELLSKNYHLENEVARLKK" CDS 369..425 /codon_start=1 /product="GCN4 peptide optimized to improve solubility while preserving binding to an scFv-GFP fusion protein (Tanenbaum et al., 2014)" /label=GCN4_v4 /translation="EELLSKNYHLENEVARLKK" CDS 441..497 /codon_start=1 /product="GCN4 peptide optimized to improve solubility while preserving binding to an scFv-GFP fusion protein (Tanenbaum et al., 2014)" /label=GCN4_v4 /translation="EELLSKNYHLENEVARLKK" CDS 513..569 /codon_start=1 /product="GCN4 peptide optimized to improve solubility while preserving binding to an scFv-GFP fusion protein (Tanenbaum et al., 2014)" /label=GCN4_v4 /translation="EELLSKNYHLENEVARLKK" CDS 585..641 /codon_start=1 /product="GCN4 peptide optimized to improve solubility while preserving binding to an scFv-GFP fusion protein (Tanenbaum et al., 2014)" /label=GCN4_v4 /translation="EELLSKNYHLENEVARLKK" CDS 657..713 /codon_start=1 /product="GCN4 peptide optimized to improve solubility while preserving binding to an scFv-GFP fusion protein (Tanenbaum et al., 2014)" /label=GCN4_v4 /translation="EELLSKNYHLENEVARLKK" CDS 729..785 /codon_start=1 /product="GCN4 peptide optimized to improve solubility while preserving binding to an scFv-GFP fusion protein (Tanenbaum et al., 2014)" /label=GCN4_v4 /translation="EELLSKNYHLENEVARLKK" 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" regulatory 1286..1295 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1292..1666 /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 1676..1900 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1907..2147 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1907..1927 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2078..2097 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2157..2190 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2227..2246) /label=T7 /note="T7 promoter, forward primer" promoter complement(2228..2246) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2333..2350) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2504..3092) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2584..2603) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3263..4123) /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 3886..3905 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4124..4228) /gene="bla" /label=AmpR promoter primer_bind 4296..4314 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4352..4374) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4474..4493 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc gaagaacttt tgagcaagaa ttatcatctt gagaacgaag 121 tggctcgtct taagaaaggt tctggcagtg gagaagaact gctttcaaag aattaccacc 181 tggaaaatga ggtagctaga ctgaaaaagg ggagcggaag tggggaggag ttgctgagca 241 aaaattatca tttggagaac gaagtagcac gactaaagaa agggtccgga tcgggtgagg 301 agttactctc gaaaaattat catctcgaaa acgaagtggc tcggctaaaa aagggcagtg 361 gttctggaga agagctatta tctaaaaact accacctcga aaatgaggtg gcacgcttaa 421 aaaagggaag tggcagtggt gaagagctac tatccaagaa ttatcatctt gagaacgagg 481 tagcgcgttt gaagaagggt tccggctcag gagaggaact gctctcgaag aactatcatc 541 ttgaaaatga ggtcgctcga ttaaaaaagg gatcgggcag tggtgaggaa ctactttcaa 601 agaattacca cctcgaaaac gaagtagctc gattaaagaa aggttcaggg tcgggtgaag 661 aattactgag taaaaattat catctggaaa atgaggtagc gagactaaaa aaggggagtg 721 gttctggcga agagttgcta tcgaaaaatt atcatcttga gaacgaagtt gctaggctca 781 aaaagtgaac 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 catggccaag ttgaccagtg ccgttccggt 1321 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc 1381 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat 1441 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg 1501 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc 1561 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga 1621 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgactgc agcgactgtg 1681 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 1741 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 1801 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 1861 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg ctcgaggagg gcctatttcc 1921 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa 1981 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct 2041 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac 2101 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgataa 2161 cttcgtatag catacattat acgaagttat ggtaccgatg cagctagccc gcggatctgc 2221 cggtctccct atagtgagtc gtattaattt cgataagcca ggttaacctg cattaatgaa 2281 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 2341 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 2401 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 2461 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 2521 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 2581 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 2641 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 2701 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 2761 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 2821 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 2881 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 2941 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 3001 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 3061 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 3121 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 3181 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 3241 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 3301 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 3361 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 3421 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 3481 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 3541 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 3601 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 3661 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 3721 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 3781 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 3841 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 3901 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 3961 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 4021 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 4081 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 4141 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 4201 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct 4261 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc 4321 gtctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg 4381 tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg 4441 gtgttggcgg gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag 4501 tgcaccatat ggacatattg tcgttagaac gcggctacaa ttaatacata accttatgta 4561 tcatacacat acg //