LOCUS Exported 3596 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (without selection) with Dronpa3. ACCESSION . VERSION . KEYWORDS pMaCTag-16 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3596) 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 3596) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3596 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..738 /codon_start=1 /product="photoswitchable Dronpa-Green3 fluorescent protein" /label=Dronpa-Green3 /translation="VSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVK EGGPLPFAYDILTTVFCYGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNAT NDITLDGDCYIYEIRFDGVNFPANGPVMQKRTVKWEPSTEKLYVRDGVLKGDINAALSL EGGGHYRCDFKTTYKAKKVVQLPDYHFVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQ AK" polyA_signal 745..866 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(782..801) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 836..855 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 873..906 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 913..1153 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 913..933 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1084..1103 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1163..1196 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1233..1252) /label=T7 /note="T7 promoter, forward primer" promoter complement(1234..1252) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1339..1356) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(1510..2098) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1590..1609) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2269..3129) /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 2892..2911 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3130..3234) /gene="bla" /label=AmpR promoter primer_bind 3302..3320 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3358..3380) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 3480..3499 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 3580..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 3580..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccgtgagtg tgattaaacc agacatgaag atcaagctgc gtatggaagg cgctgtaaat 121 ggacacccgt tcgcgattga aggagttggc cttgggaagc ctttcgaggg aaaacagagt 181 atggacctta aagtcaaaga aggcggacct ctgcctttcg cctatgacat cttgacaact 241 gtgttctgtt acggcaacag ggtattcgcc aaatacccag aaaatatagt agactatttc 301 aagcagtcgt ttcctgaggg ctactcttgg gaacgaagca tgaattacga agacgggggc 361 atttgtaacg cgacaaacga cataaccctg gatggtgact gttatatcta tgaaattcga 421 tttgatggtg tgaactttcc tgccaatggt ccagttatgc agaagaggac tgtgaaatgg 481 gagccatcca ctgagaaatt gtatgtgcgt gatggagtgc tgaagggtga tattaacgcg 541 gctctgtcgc ttgaaggagg tggccattac cgatgtgact tcaaaactac ttataaagct 601 aagaaggttg tccagttgcc agactatcac tttgtggacc accacattga gattaaaagc 661 cacgacaaag attacagtaa tgttaatctg catgagcacg ccgaagcgca ttctgagctg 721 ccgaggcagg ccaagtgaac tagtaacttg tttattgcag cttataatgg ttacaaataa 781 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 841 ttgtccaaac tcatcaatgt atcttagtcg atataacttc gtatagcata cattatacga 901 agttatgtcg aggagggcct atttcccatg attccttcat atttgcatat acgatacaag 961 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat 1021 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa 1081 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc 1141 ttgtggaaag gacgaaacac cgataacttc gtatagcata cattatacga agttatggta 1201 ccgatgcagc tagcccgcgg atctgccggt ctccctatag tgagtcgtat taatttcgat 1261 aagccaggtt aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 1321 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 1381 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 1441 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 1501 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 1561 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 1621 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 1681 ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt 1741 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 1801 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 1861 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 1921 gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 1981 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 2041 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 2101 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 2161 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 2221 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 2281 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 2341 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 2401 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 2461 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 2521 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 2581 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 2641 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 2701 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 2761 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 2821 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 2881 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 2941 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 3001 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 3061 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 3121 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 3181 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 3241 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa 3301 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct 3361 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag 3421 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc 3481 ggcatcagag cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg 3541 ctacaattaa tacataacct tatgtatcat acacatacga tttaggtgac actata //