LOCUS Exported 5581 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with 24xSunTag. ACCESSION . VERSION . KEYWORDS pMaCTag-Z21 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5581) 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 5581) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5581 /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" CDS 801..857 /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 873..929 /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 945..1001 /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 1017..1073 /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 1089..1145 /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 1161..1217 /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 1233..1289 /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 1305..1361 /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 1377..1433 /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 1449..1505 /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 1521..1577 /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 1593..1649 /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 1737..1793 /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 1803..1924 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1840..1859) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1894..1913 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 1931..1964 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 1971..2287 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2138..2273 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2200..2219 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 2294..2303 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 2300..2674 /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 2684..2908 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 2915..3155 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2915..2935 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 3086..3105 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 3165..3198 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(3235..3254) /label=T7 /note="T7 promoter, forward primer" promoter complement(3236..3254) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3341..3358) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3512..4100) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3592..3611) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4271..5131) /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 4894..4913 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5132..5236) /gene="bla" /label=AmpR promoter primer_bind 5304..5322 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(5360..5382) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 5482..5501 /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 aaaagggctc aggctcaggc gaggagttgc tctcgaaaaa ctaccacttg gaaaatgagg 841 tcgcgaggtt gaaaaagggg agcgggtcgg gcgaggagtt attgagcaaa aactatcatt 901 tagagaacga agtcgcgcgc ttaaagaaag gctcgggctc gggcgaagaa ctcttatcga 961 agaactacca cctcgaaaat gaggtcgcca ggttgaaaaa gggcagtggc agcggggagg 1021 aactcttgag caagaactac cacttggaga atgaggtcgc gagattgaag aaagggtcgg 1081 ggagcggcga ggaattgctc agcaagaatt atcatttgga gaacgaagtc gccaggctca 1141 agaaaggctc ggggtcgggg gaggaattgt tgagtaaaaa ctaccacttg gaaaatgaag 1201 tcgccaggct caaaaaaggg agtgggagcg gcgaagagtt attgagcaaa aattaccact 1261 tggagaacga agtggcaagg ctcaagaaag ggagcggcag cggggaggag ctcttatcga 1321 agaactacca cttagagaat gaagtcgccc gcttgaagaa aggctcgggg agcggggaag 1381 agctcttgag caagaactac cacttggaaa atgaggtggc gcgcttgaag aaagggagcg 1441 ggagcgggga agagttacta tctaagaatt atcatctcga gaacgaggtg gctcgactaa 1501 agaagggctc cggcagtggg gaggaactcc tgtcgaagaa ctatcatctt gaaaatgagg 1561 ttgcaagact taaaaagggg tccggatcag gtgaggaact actcagtaag aattaccacc 1621 tggaaaacga agttgcacgt ttgaagaaag gatcaggatc aggcgaagaa ctgctctcaa 1681 aagattatca tttggaaaat gaggttgcac gtttaaaaaa gggaagtggc agtggtgagg 1741 aacttctgtc gaaaaattat catctcgaga atgaagtagc ccgacttaaa aagtgaacta 1801 gtaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 1861 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 1921 cttagtcgat ataacttcgt atagcataca ttatacgaag ttatgtcgac ggtgtggaaa 1981 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 2041 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 2101 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag 2161 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc 2221 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt 2281 ttgcaaagaa ttcgccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg 2341 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt 2401 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca 2461 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct 2521 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat 2581 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa 2641 ctgcgtgcac ttcgtggccg aggagcagga ctgactgcag cgactgtgcc ttctagttgc 2701 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 2761 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 2821 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 2881 catgctgggg atgcggtggg ctctatggct cgaggagggc ctatttccca tgattccttc 2941 atatttgcat atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa 3001 cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc 3061 agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt 3121 cgatttcttg gctttatata tcttgtggaa aggacgaaac accgataact tcgtatagca 3181 tacattatac gaagttatgg taccgatgca gctagcccgc ggatctgccg gtctccctat 3241 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg 3301 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 3361 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 3421 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 3481 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 3541 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 3601 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 3661 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 3721 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 3781 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 3841 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 3901 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 3961 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 4021 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 4081 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 4141 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 4201 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 4261 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 4321 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 4381 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 4441 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 4501 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 4561 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 4621 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 4681 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 4741 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 4801 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 4861 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 4921 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 4981 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 5041 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 5101 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 5161 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 5221 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta 5281 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt 5341 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 5401 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 5461 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg 5521 acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac 5581 g //