LOCUS Exported 3846 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Expresses Protein A and Protein G IgG binding domains fused to micrococcal nuclease for purposes of CUT. ACCESSION . VERSION . KEYWORDS pAG/MNase SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 3846) AUTHORS Meers MP, Bryson TD, Henikoff JG, Henikoff S TITLE Improved CUT JOURNAL Elife. 2019 Jun 24;8. pii: 46314. doi: 10.7554/eLife.46314. PUBMED 31232687 REFERENCE 2 (bases 1 to 3846) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..3846 /organism="synthetic DNA construct" /mol_type="other DNA" CDS 365..1159 /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(419..438) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 1029..1048 /label=Neo-F /note="Neomycin resistance gene, forward primer" rep_origin 1443..2031 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 1932..1951 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 2185..2202 /label=L4440 /note="L4440 vector, forward primer" protein_bind 2319..2340 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 2355..2385 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 2393..2409 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 2398..2420 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 2417..2433 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 2417..2433 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" CDS 2447..2872 /codon_start=1 /product="immunoglobulin binding domains of Staphylocuccus aureus protein A" /label=protein A /translation="SLKDDPSQSANLLSEAKKLNESQAPKADNKFNKEQQNAFYEILHL PNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLP NLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK" CDS 2873..3082 /codon_start=1 /product="immunoglobulin binding domain of Streptoccocus protein G" /label=protein G /translation="TTYKLVINGKTLKGETTTEAVDAETAERHFKQYANDNGVDGEWTY DDATKTFTVTEKPEVIDASELTPAV" CDS 3101..3547 /codon_start=1 /product="micrococcal nuclease from Staphylococcus aureus" /label=MNase /translation="ATSTKKLHKEPATLIKAIDGDTVKLMYKGQPMTFRLLLVDTPETK HPKKGVEKYGPEASAFTKKMVENAKKIEVEFDKGQRTDKYGRGLAYIYADGKMVNEALV RQGLAKVAYVYKPNNTHEQHLRKSEAQAKKEKLNIWSEDNADSGQ" CDS 3593..3610 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 3626..3652 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" primer_bind complement(3690..3707) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(3690..3706) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(3699..3721) /label=M13/pUC Forward /note="In lacZ gene" ORIGIN 1 cgataagcta gcttcacgct gccgcaagca ctcagggcgc aagggctgct aaaggaagcg 61 gaacacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg tcagctactg 121 ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt gcagtgggct 181 tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg aattgccagc 241 tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc 301 gccaaggatc tgatggcgca ggggatcaag atctgatcaa gagacaggat gaggatcgtt 361 tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 421 attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 481 gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 541 actccaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 601 tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 661 gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 721 aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 781 tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 841 cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgcggatgcc 901 cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 961 aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 1021 ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 1081 cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 1141 tcttgacgag ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc 1201 aacctgccat cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga 1261 atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc 1321 ttcgcccacc ccaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 1381 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 1441 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 1501 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 1561 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac 1621 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 1681 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 1741 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 1801 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 1861 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 1921 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 1981 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 2041 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct 2101 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct 2161 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca 2221 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 2281 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat 2341 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc 2401 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct taaaagatga 2461 cccaagccaa agtgctaacc tattgtcaga agctaaaaag ttaaatgaat ctcaagcacc 2521 gaaagcggat aacaaattca acaaagaaca acaaaatgct ttctatgaaa tcttacattt 2581 acctaactta aacgaagaac aacgcaatgg tttcatccaa agcctaaaag atgacccaag 2641 ccaaagcgct aaccttttag cagaagctaa aaagctaaat gatgctcaag caccaaaagc 2701 tgacaacaaa ttcaacaaag aacaacaaaa tgctttctat gaaattttac atttacctaa 2761 cttaactgaa gaacaacgta acggcttcat ccaaagcctt aaagacgatc cttcagtgag 2821 caaagaaatt ttagcagaag ctaaaaagct aaacgatgct caagcaccaa aaacaactta 2881 taaattagtc atcaacggga aaacgctgaa gggtgaaacc acgacagagg ccgtagatgc 2941 ggagacagcg gagcgccact ttaagcaata cgcgaatgat aacggtgtag acggcgagtg 3001 gacctacgac gacgcgacaa agacctttac cgtcacggag aaacctgagg ttatcgacgc 3061 gtctgagttg acgccagccg tagatgacga taaagaattc gcaacttcaa ctaaaaaatt 3121 acataaagaa cctgcgactt taattaaagc gattgatggt gatacggtta aattaatgta 3181 caaaggtcaa ccaatgacat tcagactatt attggtcgac acacctgaaa caaagcatcc 3241 taaaaaaggt gtagagaaat atggtcctga agcaagtgca tttaccaaaa aaatggtaga 3301 aaatgcaaag aaaattgaag tcgaattcga caaaggtcaa agaactgata aatatggacg 3361 tgggctagcg tatatttatg ctgatggaaa aatggtaaac gaagctttag ttcgtcaagg 3421 cttggctaaa gttgcttatg tttacaaacc taacaataca catgaacaac atttaagaaa 3481 aagtgaagca caagcgaaaa aagagaaatt aaatatttgg agcgaagaca acgctgattc 3541 aggtcaaggc ggtggtggct ctgggggcgg gggctcgggt ggtgggggct cacaccatca 3601 ccatcaccat ggcggtggtg gctcttaccc atacgatgtt ccagattacg cttaatgagg 3661 atccccgggt accgagctcg aattaattca ctggccgtcg ttttacaacg tcgtgactgg 3721 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 3781 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 3841 gaatgg //