LOCUS Exported 4582 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Zeocin selection) with eGFP. ACCESSION . VERSION . KEYWORDS pMaCTag-Z05 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4582) 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 4582) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4582 /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..797 /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 81..794 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE FVTAAGITLGMDELYK" primer_bind complement(123..144) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(384..403) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 731..752 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 804..925 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(841..860) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 895..914 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 932..965 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 972..1288 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 1139..1274 /label=SV40 ori /note="SV40 origin of replication" primer_bind 1201..1220 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" regulatory 1295..1304 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1301..1675 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 1685..1909 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1916..2156 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1916..1936 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2087..2106 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 2166..2199 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(2236..2255) /label=T7 /note="T7 promoter, forward primer" promoter complement(2237..2255) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(2342..2359) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2513..3101) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2593..2612) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(3272..4132) /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 3895..3914 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(4133..4237) /gene="bla" /label=AmpR promoter primer_bind 4305..4323 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(4361..4383) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4483..4502 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 atttaggtga cactatagaa cgcggccgcc agctgaagct tcgtacgtca ggtggaggag 61 gtagtggcgg aggcggatcc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 121 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg 181 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 241 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 301 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 361 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 421 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 481 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 541 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 601 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 661 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 721 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 781 acgagctgta caagtaaact agtaacttgt ttattgcagc ttataatggt tacaaataaa 841 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 901 tgtccaaact catcaatgta tcttagtcga tataacttcg tatagcatac attatacgaa 961 gttatgtcga cggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat 1021 gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag 1081 tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat 1141 cccgccccta actccgccca gttccgccca ttctccgccc catggctgac taattttttt 1201 tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg 1261 cttttttgga ggcctaggct tttgcaaaga attcgccacc atggccaagt tgaccagtgc 1321 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct 1381 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac 1441 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg 1501 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg 1561 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc 1621 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgactgca 1681 gcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 1741 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 1801 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 1861 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc tcgaggaggg 1921 cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta gagagataat 1981 tggaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac gtagaaagta 2041 ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact atcatatgct 2101 taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga aaggacgaaa 2161 caccgataac ttcgtatagc atacattata cgaagttatg gtaccgatgc agctagcccg 2221 cggatctgcc ggtctcccta tagtgagtcg tattaatttc gataagccag gttaacctgc 2281 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 2341 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 2401 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 2461 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 2521 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 2581 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 2641 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 2701 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 2761 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 2821 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 2881 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 2941 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 3001 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 3061 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 3121 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat 3181 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 3241 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 3301 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 3361 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 3421 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 3481 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 3541 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 3601 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 3661 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 3721 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 3781 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 3841 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 3901 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 3961 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 4021 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 4081 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 4141 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 4201 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 4261 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga 4321 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc 4381 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg 4441 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg 4501 tactgagagt gcaccatatg gacatattgt cgttagaacg cggctacaat taatacataa 4561 ccttatgtat catacacata cg //