LOCUS Exported 4789 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with TagBFP. ACCESSION . VERSION . KEYWORDS pMaCTag-P02 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4789) 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 4789) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4789 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..762 /codon_start=1 /product="monomeric blue fluorescent protein" /label=TagBFP /note="mammalian codon-optimized" /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD LPSKLGHKLN" polyA_signal 769..890 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(806..825) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 860..879 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 897..930 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 937..1253 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1104..1239 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1166..1185 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 1875..2099 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 2106..2346 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2106..2126 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2277..2296 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2356..2389 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2426..2445) /label=T7 /note="T7 promoter, forward primer" promoter complement(2427..2445) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2532..2549) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2703..3291) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2783..2802) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3462..4322) /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 4085..4104 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4323..4427) /gene="bla" /label=AmpR promoter primer_bind 4495..4513 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4551..4573) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4673..4692 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 4773..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 4773..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tccagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtggac 121 aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc 181 atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctact 241 agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc cgacttcttc 301 aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc 361 gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag 421 atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac actcggctgg 481 gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag aaacgacatg 541 gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac atatagatcc 601 aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta cagactggaa 661 agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc agtggccaga 721 tactgcgacc tccctagcaa actggggcac aagcttaatt gaactagtaa cttgtttatt 781 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt 841 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta gtcgatataa 901 cttcgtatag catacattat acgaagttat gtcgacggtg tggaaagtcc ccaggctccc 961 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt 1021 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca 1081 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc 1141 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg 1201 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagaattcg 1261 ccaccatgac tgagtacaag cctactgtgc gtttagccac cagggatgat gtgccacgag 1321 ccgtgaggac actagcagcc gcctttgctg actacccagc tacaaggcac accgtggacc 1381 ctgacagaca cattgaaaga gtcacagaac ttcaggagct gtttttaact agggtgggcc 1441 tggatattgg caaagtctgg gtggccgatg atggagcagc agttgcagtg tggaccaccc 1501 cagagtctgt tgaagctgga gctgtctttg cagaaattgg accaagaatg gccgagttat 1561 ctggcagcag gctggctgct caacagcaga tggaaggatt attagctccc cacagaccca 1621 aggagcctgc ctggttcctg gcaacagttg gagtttctcc agaccatcag ggcaagggct 1681 taggatctgc tgtggtgctg cctggtgtgg aagcagctga gagagctggg gtgccagcat 1741 ttctggagac atcagctcct agaaacctgc ccttctatga acggctgggc ttcacagtga 1801 ctgctgatgt ggaggtgcct gagggcccca gaacctggtg catgacaaga aaaccaggag 1861 cctgactgca gcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt 1921 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 1981 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 2041 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 2101 tcgaggaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta 2161 gagagataat tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac 2221 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact 2281 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga 2341 aaggacgaaa caccgataac ttcgtatagc atacattata cgaagttatg gtaccgatgc 2401 agctagcccg cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag 2461 gttaacctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc 2521 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 2581 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 2641 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 2701 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 2761 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 2821 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 2881 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 2941 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 3001 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 3061 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 3121 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 3181 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 3241 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 3301 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 3361 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 3421 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 3481 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 3541 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 3601 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 3661 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 3721 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 3781 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 3841 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 3901 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 3961 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 4021 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 4081 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 4141 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 4201 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 4261 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 4321 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 4381 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 4441 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 4501 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 4561 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 4621 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 4681 gagcagattg tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat 4741 taatacataa ccttatgtat catacacata cgatttaggt gacactata //