LOCUS       pAG/MNase                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
            //gt12365.cn
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
//