LOCUS Exported 4135 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with V5. ACCESSION . VERSION . KEYWORDS pMaCTag-P31 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4135) 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 4135) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4135 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..105 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" polyA_signal 115..236 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(152..171) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 206..225 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 243..276 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 283..599 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 450..585 /label=SV40 ori /note="SV40 origin of replication" primer_bind 512..531 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 1221..1445 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1452..1692 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1452..1472 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1623..1642 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1702..1735 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1772..1791) /label=T7 /note="T7 promoter, forward primer" promoter complement(1773..1791) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1878..1895) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2049..2637) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2129..2148) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2808..3668) /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 3431..3450 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3669..3773) /gene="bla" /label=AmpR promoter primer_bind 3841..3859 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3897..3919) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4019..4038 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 4119..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 4119..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgtgaac tagtaacttg 121 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 181 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttagtcg 241 atataacttc gtatagcata cattatacga agttatgtcg acggtgtgga aagtccccag 301 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 361 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 421 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 481 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg 541 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag 601 aattcgccac catgactgag tacaagccta ctgtgcgttt agccaccagg gatgatgtgc 661 cacgagccgt gaggacacta gcagccgcct ttgctgacta cccagctaca aggcacaccg 721 tggaccctga cagacacatt gaaagagtca cagaacttca ggagctgttt ttaactaggg 781 tgggcctgga tattggcaaa gtctgggtgg ccgatgatgg agcagcagtt gcagtgtgga 841 ccaccccaga gtctgttgaa gctggagctg tctttgcaga aattggacca agaatggccg 901 agttatctgg cagcaggctg gctgctcaac agcagatgga aggattatta gctccccaca 961 gacccaagga gcctgcctgg ttcctggcaa cagttggagt ttctccagac catcagggca 1021 agggcttagg atctgctgtg gtgctgcctg gtgtggaagc agctgagaga gctggggtgc 1081 cagcatttct ggagacatca gctcctagaa acctgccctt ctatgaacgg ctgggcttca 1141 cagtgactgc tgatgtggag gtgcctgagg gccccagaac ctggtgcatg acaagaaaac 1201 caggagcctg actgcagcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 1261 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 1321 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 1381 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 1441 tatggctcga ggagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 1501 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 1561 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 1621 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 1681 tgtggaaagg acgaaacacc gataacttcg tatagcatac attatacgaa gttatggtac 1741 cgatgcagct agcccgcgga tctgccggtc tccctatagt gagtcgtatt aatttcgata 1801 agccaggtta acctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 1861 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 1921 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 1981 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 2041 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 2101 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 2161 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 2221 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 2281 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 2341 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 2401 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 2461 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 2521 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 2581 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 2641 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 2701 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 2761 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 2821 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 2881 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 2941 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 3001 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 3061 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 3121 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 3181 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 3241 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 3301 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 3361 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 3421 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 3481 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 3541 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 3601 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 3661 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 3721 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 3781 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 3841 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc 3901 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 3961 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 4021 gcatcagagc agattgtact gagagtgcac catatggaca tattgtcgtt agaacgcggc 4081 tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca ctata //