LOCUS Exported 3895 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with 1xHA. ACCESSION . VERSION . KEYWORDS pMaCTag-Z27 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3895) 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 3895) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3895 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..90 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" polyA_signal 100..221 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(137..156) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 191..210 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 228..261 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 268..584 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 435..570 /label=SV40 ori /note="SV40 origin of replication" primer_bind 497..516 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 591..600 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 597..971 /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 981..1205 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1212..1452 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1212..1232 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1383..1402 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1462..1495 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1532..1551) /label=T7 /note="T7 promoter, forward primer" promoter complement(1533..1551) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1638..1655) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1809..2397) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1889..1908) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2568..3428) /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 3191..3210 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3429..3533) /gene="bla" /label=AmpR promoter primer_bind 3601..3619 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3657..3679) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3779..3798 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 3879..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 3879..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tcctacccat acgacgtccc agactacgct tgaactagta acttgtttat tgcagcttat 121 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 181 cattctagtt gtggtttgtc caaactcatc aatgtatctt agtcgatata acttcgtata 241 gcatacatta tacgaagtta tgtcgacggt gtggaaagtc cccaggctcc ccagcaggca 301 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct 361 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc 421 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg 481 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc 541 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagaattc gccaccatgg 601 ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt 661 tctggaccga ccggctcggg ttctcccggg acttcgtgga ggacgacttc gccggtgtgg 721 tccgggacga cgtgaccctg ttcatcagcg cggtccagga ccaggtggtg ccggacaaca 781 ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg 841 tgtccacgaa cttccgggac gcctccgggc cggccatgac cgagatcggc gagcagccgt 901 gggggcggga gttcgccctg cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg 961 agcaggactg actgcagcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 1021 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 1081 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 1141 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 1201 tatggctcga ggagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 1261 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 1321 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 1381 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 1441 tgtggaaagg acgaaacacc gataacttcg tatagcatac attatacgaa gttatggtac 1501 cgatgcagct agcccgcgga tctgccggtc tccctatagt gagtcgtatt aatttcgata 1561 agccaggtta acctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 1621 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 1681 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 1741 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 1801 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 1861 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 1921 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 1981 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 2041 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 2101 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 2161 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 2221 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 2281 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 2341 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 2401 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 2461 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 2521 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 2581 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 2641 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 2701 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 2761 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 2821 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 2881 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 2941 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 3001 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 3061 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 3121 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 3181 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 3241 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 3301 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 3361 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 3421 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 3481 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 3541 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 3601 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc 3661 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 3721 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 3781 gcatcagagc agattgtact gagagtgcac catatggaca tattgtcgtt agaacgcggc 3841 tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca ctata //