LOCUS Exported 3958 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with 3xHA. ACCESSION . VERSION . KEYWORDS pMaCTag-Z28 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3958) 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 3958) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3958 /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..170 /codon_start=1 /product="three tandem HA epitope tags" /label=3xHA /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA" polyA_signal 180..301 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(217..236) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 271..290 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 308..341 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 348..664 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 515..650 /label=SV40 ori /note="SV40 origin of replication" primer_bind 577..596 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 671..680 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 677..1051 /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 1061..1285 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1292..1532 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1292..1312 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1463..1482 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1542..1575 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1612..1631) /label=T7 /note="T7 promoter, forward primer" promoter complement(1613..1631) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1718..1735) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1889..2477) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1969..1988) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2648..3508) /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 3271..3290 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3509..3613) /gene="bla" /label=AmpR promoter primer_bind 3681..3699 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3737..3759) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3859..3878 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc tacccatacg atgttcctga ctatgcgggc tatccctatg 121 acgtcccgga ctatgcagga tcctatccat atgacgttcc agattacgct tgaactagta 181 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 241 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt 301 agtcgatata acttcgtata gcatacatta tacgaagtta tgtcgacggt gtggaaagtc 361 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag 421 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 481 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 541 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 601 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 661 caaagaattc gccaccatgg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 721 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 781 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 841 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 901 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 961 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 1021 cgtgcacttc gtggccgagg agcaggactg actgcagcga ctgtgccttc tagttgccag 1081 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 1141 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt 1201 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat 1261 gctggggatg cggtgggctc tatggctcga ggagggccta tttcccatga ttccttcata 1321 tttgcatata cgatacaagg ctgttagaga gataattgga attaatttga ctgtaaacac 1381 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt 1441 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga 1501 tttcttggct ttatatatct tgtggaaagg acgaaacacc gataacttcg tatagcatac 1561 attatacgaa gttatggtac cgatgcagct agcccgcgga tctgccggtc tccctatagt 1621 gagtcgtatt aatttcgata agccaggtta acctgcatta atgaatcggc caacgcgcgg 1681 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1741 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1801 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 1861 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 1921 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 1981 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 2041 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2101 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 2161 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 2221 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 2281 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 2341 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 2401 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2461 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 2521 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 2581 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 2641 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 2701 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 2761 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag 2821 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct 2881 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt 2941 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 3001 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 3061 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 3121 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat 3181 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac 3241 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa 3301 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 3361 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 3421 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 3481 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt 3541 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa 3601 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta 3661 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg 3721 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt 3781 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc 3841 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatggaca 3901 tattgtcgtt agaacgcggc tacaattaat acataacctt atgtatcata cacatacg //