LOCUS Exported 5191 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with YFP-LID. ACCESSION . VERSION . KEYWORDS pMaCTag-P25 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5191) 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 5191) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5191 /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..794 /codon_start=1 /product="enhanced YFP" /label=EYFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(123..144) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(384..403) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 731..752 /label=EGFP-C /note="EGFP, forward primer" CDS 801..1121 /codon_start=1 /product="F36V mutant of FK506-binding protein FKBP12" /label=FKBP-V /note="binds synthetic ligands that do not bind wild-type FKBP" /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL KLE" polyA_signal 1188..1309 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1225..1244) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1279..1298 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 1316..1349 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 1356..1672 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1523..1658 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1585..1604 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 2294..2518 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 2525..2765 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2525..2545 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2696..2715 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2775..2808 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2845..2864) /label=T7 /note="T7 promoter, forward primer" promoter complement(2846..2864) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2951..2968) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(3122..3710) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3202..3221) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3881..4741) /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 4504..4523 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4742..4846) /gene="bla" /label=AmpR promoter primer_bind 4914..4932 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4970..4992) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 5092..5111 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 121 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 181 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 241 ccgtgccctg gcccaccctc gtgaccacct tcggctacgg cctgcagtgc ttcgcccgct 301 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 361 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 421 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 481 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 541 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 601 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 661 tgctgcccga caaccactac ctgagctacc agtccgccct gagcaaagac cccaacgaga 721 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 781 acgagctgta caaggaattc ggagtgcagg tggaaaccat ctccccagga gacgggcgca 841 ccttccccaa gcgcggccag acctgcgtgg tgcactacac cgggatgctt gaagatggaa 901 agaaagttga ttcctcccgg gacagaaaca agccctttaa gtttatgcta ggcaagcagg 961 aggtgatccg aggctgggaa gaaggggttg cccagatgag tgtgggtcag agagccaaac 1021 tgactatatc tccagattat gcctatggtg ccactgggca cccaggcatc atcccaccac 1081 atgccactct cgtcttcgat gtggagcttc taaaactgga aacgcgtgga gtggaggaag 1141 tggcggaggg ggtagttctc ttaaggcggc gaggtaatta gactagtaac ttgtttattg 1201 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 1261 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttag tcgatataac 1321 ttcgtatagc atacattata cgaagttatg tcgacggtgt ggaaagtccc caggctcccc 1381 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc 1441 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat 1501 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc 1561 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga 1621 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagaattcgc 1681 caccatgact gagtacaagc ctactgtgcg tttagccacc agggatgatg tgccacgagc 1741 cgtgaggaca ctagcagccg cctttgctga ctacccagct acaaggcaca ccgtggaccc 1801 tgacagacac attgaaagag tcacagaact tcaggagctg tttttaacta gggtgggcct 1861 ggatattggc aaagtctggg tggccgatga tggagcagca gttgcagtgt ggaccacccc 1921 agagtctgtt gaagctggag ctgtctttgc agaaattgga ccaagaatgg ccgagttatc 1981 tggcagcagg ctggctgctc aacagcagat ggaaggatta ttagctcccc acagacccaa 2041 ggagcctgcc tggttcctgg caacagttgg agtttctcca gaccatcagg gcaagggctt 2101 aggatctgct gtggtgctgc ctggtgtgga agcagctgag agagctgggg tgccagcatt 2161 tctggagaca tcagctccta gaaacctgcc cttctatgaa cggctgggct tcacagtgac 2221 tgctgatgtg gaggtgcctg agggccccag aacctggtgc atgacaagaa aaccaggagc 2281 ctgactgcag cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 2341 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 2401 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 2461 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct 2521 cgaggagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag 2581 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg 2641 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta 2701 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa 2761 aggacgaaac accgataact tcgtatagca tacattatac gaagttatgg taccgatgca 2821 gctagcccgc ggatctgccg gtctccctat agtgagtcgt attaatttcg ataagccagg 2881 ttaacctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct 2941 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat 3001 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga 3061 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 3121 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 3181 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 3241 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 3301 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 3361 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 3421 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 3481 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 3541 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta 3601 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 3661 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 3721 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 3781 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 3841 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 3901 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 3961 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 4021 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 4081 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 4141 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 4201 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 4261 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 4321 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 4381 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 4441 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 4501 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 4561 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 4621 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 4681 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 4741 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 4801 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 4861 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 4921 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 4981 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 5041 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 5101 agcagattgt actgagagtg caccatatgg acatattgtc gttagaacgc ggctacaatt 5161 aatacataac cttatgtatc atacacatac g //