LOCUS Exported 4135 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with V5. ACCESSION . VERSION . KEYWORDS pMaCTag-P31 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4135) 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 4135) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4135 /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..122 /codon_start=1 /product="epitope tag from simian virus 5" /label=V5 tag /translation="GKPIPNPLLGLDST" polyA_signal 132..253 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(169..188) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 223..242 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 260..293 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 300..616 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 467..602 /label=SV40 ori /note="SV40 origin of replication" primer_bind 529..548 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 1238..1462 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1469..1709 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1469..1489 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1640..1659 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1719..1752 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1789..1808) /label=T7 /note="T7 promoter, forward primer" promoter complement(1790..1808) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1895..1912) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2066..2654) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2146..2165) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2825..3685) /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 3448..3467 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3686..3790) /gene="bla" /label=AmpR promoter primer_bind 3858..3876 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3914..3936) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4036..4055 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc ggtaagccta tccctaaccc tctcctcggt ctcgattcta 121 cgtgaactag taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 181 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 241 tcaatgtatc ttagtcgata taacttcgta tagcatacat tatacgaagt tatgtcgacg 301 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 361 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 421 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac 481 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga 541 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg 601 cctaggcttt tgcaaagaat tcgccaccat gactgagtac aagcctactg tgcgtttagc 661 caccagggat gatgtgccac gagccgtgag gacactagca gccgcctttg ctgactaccc 721 agctacaagg cacaccgtgg accctgacag acacattgaa agagtcacag aacttcagga 781 gctgttttta actagggtgg gcctggatat tggcaaagtc tgggtggccg atgatggagc 841 agcagttgca gtgtggacca ccccagagtc tgttgaagct ggagctgtct ttgcagaaat 901 tggaccaaga atggccgagt tatctggcag caggctggct gctcaacagc agatggaagg 961 attattagct ccccacagac ccaaggagcc tgcctggttc ctggcaacag ttggagtttc 1021 tccagaccat cagggcaagg gcttaggatc tgctgtggtg ctgcctggtg tggaagcagc 1081 tgagagagct ggggtgccag catttctgga gacatcagct cctagaaacc tgcccttcta 1141 tgaacggctg ggcttcacag tgactgctga tgtggaggtg cctgagggcc ccagaacctg 1201 gtgcatgaca agaaaaccag gagcctgact gcagcgactg tgccttctag ttgccagcca 1261 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 1321 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 1381 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 1441 ggggatgcgg tgggctctat ggctcgagga gggcctattt cccatgattc cttcatattt 1501 gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg taaacacaaa 1561 gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt ttgcagtttt 1621 aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt atttcgattt 1681 cttggcttta tatatcttgt ggaaaggacg aaacaccgat aacttcgtat agcatacatt 1741 atacgaagtt atggtaccga tgcagctagc ccgcggatct gccggtctcc ctatagtgag 1801 tcgtattaat ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga 1861 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 1921 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 1981 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 2041 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 2101 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 2161 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 2221 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 2281 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 2341 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 2401 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 2461 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 2521 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 2581 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 2641 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 2701 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 2761 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 2821 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 2881 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 2941 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 3001 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 3061 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 3121 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 3181 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 3241 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 3301 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 3361 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 3421 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 3481 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 3541 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 3601 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 3661 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 3721 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 3781 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 3841 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg 3901 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag 3961 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg 4021 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atggacatat 4081 tgtcgttaga acgcggctac aattaataca taaccttatg tatcatacac atacg //