LOCUS Exported 5035 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with miRFP670. ACCESSION . VERSION . KEYWORDS pMaCTag-P15 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5035) 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 5035) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5035 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..1008 /codon_start=1 /product="bright monomeric near-infrared fluorescent protein engineered from a bacterial phytochrome, with an emission maximum of 670 nm (Shcherbakova et al., 2016)" /label=miRFP670 /label=ORF frame 1 /translation="VAGHASGSPAFGTASHSNCEHEEIHLAGSIQPHGALLVVSEHDHR VIQASANAAEFLNLGSVLGVPLAEIDGDLLIKILPHLDPTAEGMPVAVRCRIGNPSTEY CGLMHRPPEGGLIIELERAGPSIDLSGTLAPALERIRTAGSLRALCDDTVLLFQQCTGY DRVMVYRFDEQGHGLVFSECHVPGLESYFGNRYPSSTVPQMARQLYVRQRVRVLVDVTY QPVPLEPRLSPLTGRDLDMSGCFLRSMSPCHLQFLKDMGVRATLAVSLVVGGKLWGLVV CHHYLPRFIRFELRAICKRLAERIATRITALES" polyA_signal 1015..1136 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(1052..1071) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 1106..1125 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 1143..1176 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 1183..1499 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1350..1485 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1412..1431 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 2121..2345 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 2352..2592 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2352..2372 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2523..2542 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2602..2635 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2672..2691) /label=T7 /note="T7 promoter, forward primer" promoter complement(2673..2691) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2778..2795) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2949..3537) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(3029..3048) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3708..4568) /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 4331..4350 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4569..4673) /gene="bla" /label=AmpR promoter primer_bind 4741..4759 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4797..4819) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4919..4938 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 5019..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 5019..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tctgtagcag gtcatgcctc tggcagcccc gcattcggga ccgcctctca ttcgaattgc 121 gaacatgaag agatccacct cgccggctcg atccagccgc atggcgcgct tctggtcgtc 181 agcgaacatg atcatcgcgt catccaggcc agcgccaacg ccgcggaatt tctgaatctc 241 ggaagcgtac tcggcgttcc gctcgccgag atcgacggcg atctgttgat caagatcctg 301 ccgcatctcg atcccaccgc cgaaggcatg ccggtcgcgg tgcgctgccg gatcggcaat 361 ccctctacgg agtactgcgg tctgatgcat cggcctccgg aaggcgggct gatcatcgaa 421 ctcgaacgtg ccggcccgtc gatcgatctg tcaggcacgc tggcgccggc gctggagcgg 481 atccgcacgg cgggttcact gcgcgcgctg tgcgatgaca ccgtgctgct gtttcagcag 541 tgcaccggct acgaccgggt gatggtgtat cgtttcgatg agcaaggcca cggcctggta 601 ttctccgagt gccatgtgcc tgggctcgaa tcctatttcg gcaaccgcta tccgtcgtcg 661 actgtcccgc agatggcgcg gcagctgtac gtgcggcagc gcgtccgcgt gctggtcgac 721 gtcacctatc agccggtgcc gctggagccg cggctgtcgc cgctgaccgg gcgcgatctc 781 gacatgtcgg gctgcttcct gcgctcgatg tcgccgtgcc atctgcagtt cctgaaggac 841 atgggcgtgc gcgccaccct ggcggtgtcg ctggtggtcg gcggcaagct gtggggcctg 901 gttgtctgtc accattatct gccgcgcttc atccgtttcg agctgcgggc gatctgcaaa 961 cggctcgccg aaaggatcgc gacgcggatc accgcgcttg agagctgaac tagtaacttg 1021 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 1081 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttagtcg 1141 atataacttc gtatagcata cattatacga agttatgtcg acggtgtgga aagtccccag 1201 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 1261 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 1321 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 1381 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg 1441 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag 1501 aattcgccac catgactgag tacaagccta ctgtgcgttt agccaccagg gatgatgtgc 1561 cacgagccgt gaggacacta gcagccgcct ttgctgacta cccagctaca aggcacaccg 1621 tggaccctga cagacacatt gaaagagtca cagaacttca ggagctgttt ttaactaggg 1681 tgggcctgga tattggcaaa gtctgggtgg ccgatgatgg agcagcagtt gcagtgtgga 1741 ccaccccaga gtctgttgaa gctggagctg tctttgcaga aattggacca agaatggccg 1801 agttatctgg cagcaggctg gctgctcaac agcagatgga aggattatta gctccccaca 1861 gacccaagga gcctgcctgg ttcctggcaa cagttggagt ttctccagac catcagggca 1921 agggcttagg atctgctgtg gtgctgcctg gtgtggaagc agctgagaga gctggggtgc 1981 cagcatttct ggagacatca gctcctagaa acctgccctt ctatgaacgg ctgggcttca 2041 cagtgactgc tgatgtggag gtgcctgagg gccccagaac ctggtgcatg acaagaaaac 2101 caggagcctg actgcagcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc 2161 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga 2221 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca 2281 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc 2341 tatggctcga ggagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 2401 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 2461 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 2521 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 2581 tgtggaaagg acgaaacacc gataacttcg tatagcatac attatacgaa gttatggtac 2641 cgatgcagct agcccgcgga tctgccggtc tccctatagt gagtcgtatt aatttcgata 2701 agccaggtta acctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 2761 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 2821 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 2881 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 2941 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 3001 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 3061 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 3121 tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc 3181 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 3241 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 3301 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 3361 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 3421 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 3481 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 3541 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 3601 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 3661 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 3721 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 3781 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 3841 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 3901 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 3961 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 4021 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 4081 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 4141 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 4201 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 4261 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 4321 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 4381 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 4441 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 4501 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 4561 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 4621 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 4681 ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt aacctataaa 4741 aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc 4801 tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 4861 caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 4921 gcatcagagc agattgtact gagagtgcac catatggaca tattgtcgtt agaacgcggc 4981 tacaattaat acataacctt atgtatcata cacatacgat ttaggtgaca ctata //