LOCUS Exported 5806 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with 24xSunTag. ACCESSION . VERSION . KEYWORDS pMaCTag-P21 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5806) 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 5806) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5806 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..120 /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 136..192 /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 208..264 /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 280..336 /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 352..408 /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 424..480 /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 496..552 /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 568..624 /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 640..696 /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 712..768 /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 784..840 /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 856..912 /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 928..984 /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 1000..1056 /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 1072..1128 /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 1144..1200 /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 1216..1272 /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 1288..1344 /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 1360..1416 /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 1432..1488 /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 1504..1560 /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 1576..1632 /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 1720..1776 /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 1786..1907 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1823..1842) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1877..1896 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 1914..1947 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 1954..2270 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2121..2256 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2183..2202 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 2892..3116 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 3123..3363 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 3123..3143 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 3294..3313 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 3373..3406 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(3443..3462) /label=T7 /note="T7 promoter, forward primer" promoter complement(3444..3462) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3549..3566) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3720..4308) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3800..3819) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(4479..5339) /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 5102..5121 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(5340..5444) /gene="bla" /label=AmpR promoter primer_bind 5512..5530 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(5568..5590) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 5690..5709 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 5790..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 5790..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccgaagaac ttttgagcaa gaattatcat cttgagaacg aagtggctcg tcttaagaaa 121 ggttctggca gtggagaaga actgctttca aagaattacc acctggaaaa tgaggtagct 181 agactgaaaa aggggagcgg aagtggggag gagttgctga gcaaaaatta tcatttggag 241 aacgaagtag cacgactaaa gaaagggtcc ggatcgggtg aggagttact ctcgaaaaat 301 tatcatctcg aaaacgaagt ggctcggcta aaaaagggca gtggttctgg agaagagcta 361 ttatctaaaa actaccacct cgaaaatgag gtggcacgct taaaaaaggg aagtggcagt 421 ggtgaagagc tactatccaa gaattatcat cttgagaacg aggtagcgcg tttgaagaag 481 ggttccggct caggagagga actgctctcg aagaactatc atcttgaaaa tgaggtcgct 541 cgattaaaaa agggatcggg cagtggtgag gaactacttt caaagaatta ccacctcgaa 601 aacgaagtag ctcgattaaa gaaaggttca gggtcgggtg aagaattact gagtaaaaat 661 tatcatctgg aaaatgaggt agcgagacta aaaaagggga gtggttctgg cgaagagttg 721 ctatcgaaaa attatcatct tgagaacgaa gttgctaggc tcaaaaaggg ctcaggctca 781 ggcgaggagt tgctctcgaa aaactaccac ttggaaaatg aggtcgcgag gttgaaaaag 841 gggagcgggt cgggcgagga gttattgagc aaaaactatc atttagagaa cgaagtcgcg 901 cgcttaaaga aaggctcggg ctcgggcgaa gaactcttat cgaagaacta ccacctcgaa 961 aatgaggtcg ccaggttgaa aaagggcagt ggcagcgggg aggaactctt gagcaagaac 1021 taccacttgg agaatgaggt cgcgagattg aagaaagggt cggggagcgg cgaggaattg 1081 ctcagcaaga attatcattt ggagaacgaa gtcgccaggc tcaagaaagg ctcggggtcg 1141 ggggaggaat tgttgagtaa aaactaccac ttggaaaatg aagtcgccag gctcaaaaaa 1201 gggagtggga gcggcgaaga gttattgagc aaaaattacc acttggagaa cgaagtggca 1261 aggctcaaga aagggagcgg cagcggggag gagctcttat cgaagaacta ccacttagag 1321 aatgaagtcg cccgcttgaa gaaaggctcg gggagcgggg aagagctctt gagcaagaac 1381 taccacttgg aaaatgaggt ggcgcgcttg aagaaaggga gcgggagcgg ggaagagtta 1441 ctatctaaga attatcatct cgagaacgag gtggctcgac taaagaaggg ctccggcagt 1501 ggggaggaac tcctgtcgaa gaactatcat cttgaaaatg aggttgcaag acttaaaaag 1561 gggtccggat caggtgagga actactcagt aagaattacc acctggaaaa cgaagttgca 1621 cgtttgaaga aaggatcagg atcaggcgaa gaactgctct caaaagatta tcatttggaa 1681 aatgaggttg cacgtttaaa aaagggaagt ggcagtggtg aggaacttct gtcgaaaaat 1741 tatcatctcg agaatgaagt agcccgactt aaaaagtgaa ctagtaactt gtttattgca 1801 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 1861 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttagtc gatataactt 1921 cgtatagcat acattatacg aagttatgtc gacggtgtgg aaagtcccca ggctccccag 1981 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc 2041 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag 2101 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc 2161 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc 2221 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gaattcgcca 2281 ccatgactga gtacaagcct actgtgcgtt tagccaccag ggatgatgtg ccacgagccg 2341 tgaggacact agcagccgcc tttgctgact acccagctac aaggcacacc gtggaccctg 2401 acagacacat tgaaagagtc acagaacttc aggagctgtt tttaactagg gtgggcctgg 2461 atattggcaa agtctgggtg gccgatgatg gagcagcagt tgcagtgtgg accaccccag 2521 agtctgttga agctggagct gtctttgcag aaattggacc aagaatggcc gagttatctg 2581 gcagcaggct ggctgctcaa cagcagatgg aaggattatt agctccccac agacccaagg 2641 agcctgcctg gttcctggca acagttggag tttctccaga ccatcagggc aagggcttag 2701 gatctgctgt ggtgctgcct ggtgtggaag cagctgagag agctggggtg ccagcatttc 2761 tggagacatc agctcctaga aacctgccct tctatgaacg gctgggcttc acagtgactg 2821 ctgatgtgga ggtgcctgag ggccccagaa cctggtgcat gacaagaaaa ccaggagcct 2881 gactgcagcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 2941 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 3001 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 3061 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggctcg 3121 aggagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag 3181 agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta 3241 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc 3301 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag 3361 gacgaaacac cgataacttc gtatagcata cattatacga agttatggta ccgatgcagc 3421 tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt 3481 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 3541 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 3601 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 3661 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3721 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3781 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 3841 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 3901 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 3961 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 4021 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 4081 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 4141 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 4201 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 4261 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 4321 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 4381 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 4441 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 4501 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 4561 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 4621 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 4681 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 4741 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 4801 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 4861 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 4921 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 4981 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 5041 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 5101 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 5161 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 5221 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 5281 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 5341 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 5401 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 5461 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 5521 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 5581 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 5641 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag 5701 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa 5761 tacataacct tatgtatcat acacatacga tttaggtgac actata //