LOCUS Exported 4159 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with 3xFLAG. ACCESSION . VERSION . KEYWORDS pMaCTag-P34 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4159) 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 4159) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4159 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..129 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" polyA_signal 139..260 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(176..195) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 230..249 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 267..300 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 307..623 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 474..609 /label=SV40 ori /note="SV40 origin of replication" primer_bind 536..555 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 1245..1469 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1476..1716 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1476..1496 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1647..1666 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1726..1759 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1796..1815) /label=T7 /note="T7 promoter, forward primer" promoter complement(1797..1815) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1902..1919) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2073..2661) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2153..2172) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2832..3692) /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 3455..3474 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3693..3797) /gene="bla" /label=AmpR promoter primer_bind 3865..3883 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3921..3943) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4043..4062 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 4143..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 4143..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccgactaca aagaccatga cggtgattat aaagatcatg atatcgatta caaggatgac 121 gatgacaagt gaactagtaa cttgtttatt gcagcttata atggttacaa ataaagcaat 181 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 241 aaactcatca atgtatctta gtcgatataa cttcgtatag catacattat acgaagttat 301 gtcgacggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 361 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 421 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc 481 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt 541 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt 601 ttggaggcct aggcttttgc aaagaattcg ccaccatgac tgagtacaag cctactgtgc 661 gtttagccac cagggatgat gtgccacgag ccgtgaggac actagcagcc gcctttgctg 721 actacccagc tacaaggcac accgtggacc ctgacagaca cattgaaaga gtcacagaac 781 ttcaggagct gtttttaact agggtgggcc tggatattgg caaagtctgg gtggccgatg 841 atggagcagc agttgcagtg tggaccaccc cagagtctgt tgaagctgga gctgtctttg 901 cagaaattgg accaagaatg gccgagttat ctggcagcag gctggctgct caacagcaga 961 tggaaggatt attagctccc cacagaccca aggagcctgc ctggttcctg gcaacagttg 1021 gagtttctcc agaccatcag ggcaagggct taggatctgc tgtggtgctg cctggtgtgg 1081 aagcagctga gagagctggg gtgccagcat ttctggagac atcagctcct agaaacctgc 1141 ccttctatga acggctgggc ttcacagtga ctgctgatgt ggaggtgcct gagggcccca 1201 gaacctggtg catgacaaga aaaccaggag cctgactgca gcgactgtgc cttctagttg 1261 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc 1321 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc 1381 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag 1441 gcatgctggg gatgcggtgg gctctatggc tcgaggaggg cctatttccc atgattcctt 1501 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa 1561 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg 1621 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt 1681 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccgataac ttcgtatagc 1741 atacattata cgaagttatg gtaccgatgc agctagcccg cggatctgcc ggtctcccta 1801 tagtgagtcg tattaatttc gataagccag gttaacctgc attaatgaat cggccaacgc 1861 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg 1921 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta 1981 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc 2041 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 2101 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 2161 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 2221 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt 2281 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 2341 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 2401 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 2461 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta 2521 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 2581 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 2641 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 2701 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 2761 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 2821 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 2881 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 2941 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta 3001 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 3061 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 3121 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 3181 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 3241 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 3301 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 3361 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 3421 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 3481 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 3541 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 3601 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 3661 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 3721 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 3781 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt 3841 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt 3901 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt 3961 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg 4021 tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg 4081 gacatattgt cgttagaacg cggctacaat taatacataa ccttatgtat catacacata 4141 cgatttaggt gacactata //