LOCUS Exported 4117 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Template for C-terminal PCR tagging of mammalian genes (Puromycin selection) with Strep-II. ACCESSION . VERSION . KEYWORDS pMaCTag-P29 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 4117) 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 4117) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..4117 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 64..87 /codon_start=1 /product="peptide that binds Strep-Tactin(R), an engineered form of streptavidin" /label=Strep-Tag II /translation="WSHPQFEK" polyA_signal 97..218 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(134..153) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 188..207 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" protein_bind 225..258 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." promoter 265..581 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 432..567 /label=SV40 ori /note="SV40 origin of replication" primer_bind 494..513 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" polyA_signal 1203..1427 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 1434..1674 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1434..1454 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1605..1624 /label=LKO.1 5' /note="Human U6 promoter, forward primer" protein_bind 1684..1717 /label=loxP /bound_moiety="Cre recombinase" /note="Cre-mediated recombination occurs in the 8-bp core sequence (GCATACAT)." primer_bind complement(1754..1773) /label=T7 /note="T7 promoter, forward primer" promoter complement(1755..1773) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(1860..1877) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(2031..2619) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(2111..2130) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(2790..3650) /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 3413..3432 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(3651..3755) /gene="bla" /label=AmpR promoter primer_bind 3823..3841 /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" primer_bind complement(3879..3901) /label=pGEX 3' /note="pGEX vectors, reverse primer" primer_bind 4001..4020 /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" promoter 4101..2 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 4101..1 /label=SP6 /note="SP6 promoter, forward primer" ORIGIN 1 gaacgcggcc gccagctgaa gcttcgtacg tcaggtggag gaggtagtgg cggaggcgga 61 tcctggagcc acccccagtt cgagaagtga actagtaact tgtttattgc agcttataat 121 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 181 tctagttgtg gtttgtccaa actcatcaat gtatcttagt cgatataact tcgtatagca 241 tacattatac gaagttatgt cgacggtgtg gaaagtcccc aggctcccca gcaggcagaa 301 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc 361 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc 421 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct 481 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga 541 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agaattcgcc accatgactg 601 agtacaagcc tactgtgcgt ttagccacca gggatgatgt gccacgagcc gtgaggacac 661 tagcagccgc ctttgctgac tacccagcta caaggcacac cgtggaccct gacagacaca 721 ttgaaagagt cacagaactt caggagctgt ttttaactag ggtgggcctg gatattggca 781 aagtctgggt ggccgatgat ggagcagcag ttgcagtgtg gaccacccca gagtctgttg 841 aagctggagc tgtctttgca gaaattggac caagaatggc cgagttatct ggcagcaggc 901 tggctgctca acagcagatg gaaggattat tagctcccca cagacccaag gagcctgcct 961 ggttcctggc aacagttgga gtttctccag accatcaggg caagggctta ggatctgctg 1021 tggtgctgcc tggtgtggaa gcagctgaga gagctggggt gccagcattt ctggagacat 1081 cagctcctag aaacctgccc ttctatgaac ggctgggctt cacagtgact gctgatgtgg 1141 aggtgcctga gggccccaga acctggtgca tgacaagaaa accaggagcc tgactgcagc 1201 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac 1261 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg 1321 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga 1381 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctc gaggagggcc 1441 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg 1501 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat 1561 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta 1621 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca 1681 ccgataactt cgtatagcat acattatacg aagttatggt accgatgcag ctagcccgcg 1741 gatctgccgg tctccctata gtgagtcgta ttaatttcga taagccaggt taacctgcat 1801 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 1861 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 1921 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 1981 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 2041 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 2101 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 2161 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 2221 tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 2281 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 2341 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 2401 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 2461 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 2521 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 2581 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 2641 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 2701 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 2761 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 2821 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 2881 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 2941 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 3001 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 3061 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 3121 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 3181 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 3241 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 3301 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 3361 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 3421 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 3481 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 3541 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 3601 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 3661 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 3721 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 3781 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 3841 ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg 3901 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg 3961 tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta 4021 ctgagagtgc accatatgga catattgtcg ttagaacgcg gctacaatta atacataacc 4081 ttatgtatca tacacatacg atttaggtga cactata //