LOCUS Exported 4531 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with mKeima8.5. ACCESSION . VERSION . KEYWORDS pMaCTag-Z18 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4531) 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 4531) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4531 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..726 /codon_start=1 /product="human codon-optimized monomeric red fluorescent protein with a large Stokes shift (Guan et al. 2015)" /label=hmKeima8.5 /translation="VSVIAEHMTYKVYMSGTVNGHYFEVEGDGEGKPYEGEQTVKLTVT KGGPLPFAWHILSPQLQYGSIPFTKYPADIPDYFKQSFPEGYTWVRSMNFEDGAVCTVS NDSSIRGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERMFARDGMLIGNDHMALKL EGGGHYLCEFKSAYKARKPVRMPGLYEIDRKLDVTSHNRDYTSVEQREIAIARHPLLG" polyA_signal 736..857 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(773..792) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 827..846 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 864..897 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 904..1220 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1071..1206 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1133..1152 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 1227..1236 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1233..1607 /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 1617..1841 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1848..2088 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1848..1868 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2019..2038 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2098..2131 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2168..2187) /label=T7 /note="T7 promoter, forward primer" promoter complement(2169..2187) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2274..2291) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2445..3033) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2525..2544) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3204..4064) /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 3827..3846 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4065..4169) /gene="bla" /label=AmpR promoter primer_bind 4237..4255 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4293..4315) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4415..4434 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 4515..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 4515..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccgtgagcg tgatcgccga gcacatgacc tacaaggtgt acatgagcgg caccgtgaac 121 ggccactact tcgaggtgga gggcgacggc gagggcaagc cctacgaggg cgagcagacc 181 gtgaagctga ccgtgaccaa gggtggcccc ctgcccttcg cctggcacat cctgagcccc 241 cagctccagt acggcagcat ccccttcacc aagtaccccg ccgacatccc cgactacttc 301 aagcagagct tccccgaggg ctacacctgg gtgcgcagca tgaacttcga ggacggcgcc 361 gtgtgcaccg tgagcaacga cagcagcatc cgcggcaact gcttcatcta caacgtgaag 421 atcagcggcg agaacttccc ccccaacggc cccgtgatgc agaagaagac ccagggctgg 481 gagcccagca ccgagcgcat gttcgcccgc gacggaatgc tgatcggcaa cgaccacatg 541 gccctgaagc tggagggcgg cggccactac ctgtgcgagt tcaagagcgc ctacaaggcc 601 cgcaagcccg tgaggatgcc cggcctgtac gagatcgacc gcaagctgga cgtgaccagc 661 cacaaccgcg actacaccag cgtggagcag cgcgagatcg ccatcgcccg ccaccccctg 721 ctgggctgaa ctagtaactt gtttattgca gcttataatg gttacaaata aagcaatagc 781 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 841 ctcatcaatg tatcttagtc gatataactt cgtatagcat acattatacg aagttatgtc 901 gacggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 961 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa 1021 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc 1081 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg 1141 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg 1201 gaggcctagg cttttgcaaa gaattcgcca ccatggccaa gttgaccagt gccgttccgg 1261 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct 1321 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca 1381 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg 1441 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct 1501 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg 1561 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgactg cagcgactgt 1621 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 1681 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 1741 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 1801 agacaatagc aggcatgctg gggatgcggt gggctctatg gctcgaggag ggcctatttc 1861 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta 1921 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc 1981 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa 2041 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgata 2101 acttcgtata gcatacatta tacgaagtta tggtaccgat gcagctagcc cgcggatctg 2161 ccggtctccc tatagtgagt cgtattaatt tcgataagcc aggttaacct gcattaatga 2221 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 2281 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 2341 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 2401 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 2461 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 2521 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 2581 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa 2641 tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 2701 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 2761 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 2821 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 2881 agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 2941 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 3001 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 3061 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 3121 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 3181 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 3241 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 3301 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 3361 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 3421 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 3481 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 3541 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 3601 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 3661 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 3721 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 3781 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 3841 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 3901 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 3961 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 4021 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 4081 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 4141 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 4201 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 4261 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 4321 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 4381 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga 4441 gtgcaccata tggacatatt gtcgttagaa cgcggctaca attaatacat aaccttatgt 4501 atcatacaca tacgatttag gtgacactat a //