LOCUS Exported 4540 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with Dronpa3. ACCESSION . VERSION . KEYWORDS pMaCTag-Z16 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4540) 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 4540) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4540 /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..755 /codon_start=1 /product="photoswitchable Dronpa-Green3 fluorescent protein" /label=Dronpa-Green3 /translation="VSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVK EGGPLPFAYDILTTVFCYGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNAT NDITLDGDCYIYEIRFDGVNFPANGPVMQKRTVKWEPSTEKLYVRDGVLKGDINAALSL EGGGHYRCDFKTTYKAKKVVQLPDYHFVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQ AK" polyA_signal 762..883 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(799..818) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 853..872 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 890..923 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 930..1246 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1097..1232 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1159..1178 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 1253..1262 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1259..1633 /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 1643..1867 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1874..2114 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1874..1894 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2045..2064 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2124..2157 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2194..2213) /label=T7 /note="T7 promoter, forward primer" promoter complement(2195..2213) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2300..2317) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2471..3059) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2551..2570) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3230..4090) /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 3853..3872 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4091..4195) /gene="bla" /label=AmpR promoter primer_bind 4263..4281 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4319..4341) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4441..4460 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc gtgagtgtga ttaaaccaga catgaagatc aagctgcgta 121 tggaaggcgc tgtaaatgga cacccgttcg cgattgaagg agttggcctt gggaagcctt 181 tcgagggaaa acagagtatg gaccttaaag tcaaagaagg cggacctctg cctttcgcct 241 atgacatctt gacaactgtg ttctgttacg gcaacagggt attcgccaaa tacccagaaa 301 atatagtaga ctatttcaag cagtcgtttc ctgagggcta ctcttgggaa cgaagcatga 361 attacgaaga cgggggcatt tgtaacgcga caaacgacat aaccctggat ggtgactgtt 421 atatctatga aattcgattt gatggtgtga actttcctgc caatggtcca gttatgcaga 481 agaggactgt gaaatgggag ccatccactg agaaattgta tgtgcgtgat ggagtgctga 541 agggtgatat taacgcggct ctgtcgcttg aaggaggtgg ccattaccga tgtgacttca 601 aaactactta taaagctaag aaggttgtcc agttgccaga ctatcacttt gtggaccacc 661 acattgagat taaaagccac gacaaagatt acagtaatgt taatctgcat gagcacgccg 721 aagcgcattc tgagctgccg aggcaggcca agtgaactag taacttgttt attgcagctt 781 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 841 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttagtcgata taacttcgta 901 tagcatacat tatacgaagt tatgtcgacg gtgtggaaag tccccaggct ccccagcagg 961 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg 1021 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc 1081 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca 1141 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt 1201 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagaat tcgccaccat 1261 ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga 1321 gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt 1381 ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa 1441 caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt 1501 cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc 1561 gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga 1621 ggagcaggac tgactgcagc gactgtgcct tctagttgcc agccatctgt tgtttgcccc 1681 tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat 1741 gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg 1801 caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc 1861 tctatggctc gaggagggcc tatttcccat gattccttca tatttgcata tacgatacaa 1921 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa 1981 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa 2041 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat 2101 cttgtggaaa ggacgaaaca ccgataactt cgtatagcat acattatacg aagttatggt 2161 accgatgcag ctagcccgcg gatctgccgg tctccctata gtgagtcgta ttaatttcga 2221 taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 2281 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 2341 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 2401 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 2461 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 2521 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 2581 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 2641 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 2701 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 2761 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 2821 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 2881 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 2941 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 3001 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 3061 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 3121 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 3181 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 3241 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 3301 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 3361 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 3421 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 3481 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 3541 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 3601 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 3661 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 3721 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 3781 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 3841 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 3901 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 3961 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 4021 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 4081 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 4141 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 4201 ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata 4261 aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc 4321 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca 4381 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 4441 cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg ttagaacgcg 4501 gctacaatta atacataacc ttatgtatca tacacatacg //