LOCUS Exported 4549 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with mClover3. ACCESSION . VERSION . KEYWORDS pMaCTag-Z08 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4549) 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 4549) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4549 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(106..127) /label=EGFP-N /note="EGFP, reverse primer" primer_bind 714..735 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 754..875 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(791..810) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 845..864 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 882..915 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 922..1238 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1089..1224 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1151..1170 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 1245..1254 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1251..1625 /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 1635..1859 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1866..2106 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1866..1886 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2037..2056 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2116..2149 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2186..2205) /label=T7 /note="T7 promoter, forward primer" promoter complement(2187..2205) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2292..2309) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2463..3051) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2543..2562) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3222..4082) /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 3845..3864 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4083..4187) /gene="bla" /label=AmpR promoter primer_bind 4255..4273 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4311..4333) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4433..4452 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 4533..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 4533..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccgtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 121 ggcgacgtaa acggccacaa gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac 181 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 241 ctcgtgacca ccttcggcta cggcgtggcc tgcttcagcc gctaccccga ccacatgaag 301 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctct 361 ttcaaggacg acggtaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg 421 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 481 aagctggagt acaacttcaa cagccactac gtctatatca cggccgacaa gcagaagaac 541 tgcatcaagg ctaacttcaa gatccgccac aacgttgagg acggcagcgt gcagctcgcc 601 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 661 tacctgagcc atcagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc 721 ctgctggagt tcgtgaccgc cgcctgaact agtaacttgt ttattgcagc ttataatggt 781 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct 841 agttgtggtt tgtccaaact catcaatgta tcttagtcga tataacttcg tatagcatac 901 attatacgaa gttatgtcga cggtgtggaa agtccccagg ctccccagca ggcagaagta 961 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag 1021 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa 1081 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac 1141 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt 1201 agtgaggagg cttttttgga ggcctaggct tttgcaaaga attcgccacc atggccaagt 1261 tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga 1321 ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg 1381 acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg 1441 cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca 1501 cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc 1561 gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg 1621 actgactgca gcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt 1681 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 1741 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 1801 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 1861 tcgaggaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta 1921 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac 1981 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact 2041 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga 2101 aaggacgaaa caccgataac ttcgtatagc atacattata cgaagttatg gtaccgatgc 2161 agctagcccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag 2221 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 2281 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 2341 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 2401 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 2461 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 2521 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 2581 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 2641 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 2701 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 2761 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 2821 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 2881 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 2941 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 3001 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 3061 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 3121 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 3181 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 3241 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 3301 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 3361 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 3421 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 3481 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 3541 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 3601 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 3661 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 3721 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 3781 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 3841 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 3901 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 3961 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 4021 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 4081 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 4141 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 4201 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 4261 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 4321 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 4381 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 4441 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat 4501 taatacataa ccttatgtat catacacata cgatttaggt gacactata //