LOCUS Exported 4807 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with eGFP. ACCESSION . VERSION . KEYWORDS pMaCTag-P05 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4807) 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 4807) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4807 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..780 /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" CDS 64..777 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(106..127) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(367..386) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 714..735 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 787..908 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(824..843) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 878..897 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 915..948 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 955..1271 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1122..1257 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1184..1203 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 1893..2117 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 2124..2364 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 2124..2144 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2295..2314 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2374..2407 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2444..2463) /label=T7 /note="T7 promoter, forward primer" promoter complement(2445..2463) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2550..2567) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2721..3309) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2801..2820) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3480..4340) /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 4103..4122 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4341..4445) /gene="bla" /label=AmpR promoter primer_bind 4513..4531 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4569..4591) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4691..4710 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 4791..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 4791..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 gttcagcgtg tccggcgagg gcgagggcga tgccacctac 181 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 241 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 301 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 361 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 421 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 481 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 541 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 601 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 661 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 721 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 781 actagtaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 841 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 901 gtatcttagt cgatataact tcgtatagca tacattatac gaagttatgt cgacggtgtg 961 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 1021 caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 1081 tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc 1141 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg 1201 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag 1261 gcttttgcaa agaattcgcc accatgactg agtacaagcc tactgtgcgt ttagccacca 1321 gggatgatgt gccacgagcc gtgaggacac tagcagccgc ctttgctgac tacccagcta 1381 caaggcacac cgtggaccct gacagacaca ttgaaagagt cacagaactt caggagctgt 1441 ttttaactag ggtgggcctg gatattggca aagtctgggt ggccgatgat ggagcagcag 1501 ttgcagtgtg gaccacccca gagtctgttg aagctggagc tgtctttgca gaaattggac 1561 caagaatggc cgagttatct ggcagcaggc tggctgctca acagcagatg gaaggattat 1621 tagctcccca cagacccaag gagcctgcct ggttcctggc aacagttgga gtttctccag 1681 accatcaggg caagggctta ggatctgctg tggtgctgcc tggtgtggaa gcagctgaga 1741 gagctggggt gccagcattt ctggagacat cagctcctag aaacctgccc ttctatgaac 1801 ggctgggctt cacagtgact gctgatgtgg aggtgcctga gggccccaga acctggtgca 1861 tgacaagaaa accaggagcc tgactgcagc gactgtgcct tctagttgcc agccatctgt 1921 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 1981 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 2041 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 2101 tgcggtgggc tctatggctc gaggagggcc tatttcccat gattccttca tatttgcata 2161 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat 2221 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat 2281 tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg 2341 ctttatatat cttgtggaaa ggacgaaaca ccgataactt cgtatagcat acattatacg 2401 aagttatggt accgatgcag ctagcccgcg gatctgccgg tctccctata gtgagtcgta 2461 ttaatttcga taagccaggt taacctgcat taatgaatcg gccaacgcgc ggggagaggc 2521 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 2581 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 2641 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 2701 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 2761 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 2821 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 2881 gcctttctcc cttcgggaag cgtggcgctt tctcaatgct cacgctgtag gtatctcagt 2941 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 3001 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 3061 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 3121 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 3181 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 3241 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 3301 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 3361 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 3421 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 3481 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 3541 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 3601 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 3661 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 3721 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 3781 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 3841 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 3901 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 3961 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 4021 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 4081 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 4141 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 4201 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 4261 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 4321 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 4381 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 4441 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca 4501 ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac 4561 ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat 4621 gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg 4681 cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgga catattgtcg 4741 ttagaacgcg gctacaatta atacataacc ttatgtatca tacacatacg atttaggtga 4801 cactata //