LOCUS       NmeN1029A_Cas9          8180 bp    ds-DNA  circular SYN 23-NOV-2025
DEFINITION  Protein2PAM NmeN1029A_Cas9 plasmid.
ACCESSION   NmeN1029A_Cas9
VERSION     NmeN1029A_Cas9
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
COMMENT     Imported using the Genbank importer. File name: ABE8.20-m nickase
            only chassis.gb
FEATURES             Location/Qualifiers
     rep_origin      complement(435..1023)
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /direction=LEFT
     CDS             1769..2629
                     /label="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin,
                     andrelated antibiotics"
                     /gene="bla"
                     /product="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW*"
     enhancer        3199..3502
                     /label="CMV enhancer"
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3504..3706
                     /label="CMV promoter"
                     /note="human cytomegalovirus (CMV) immediate earlypromoter"
     CDS             7077..7097
                     /label="SV40 NLS"
                     /product="nuclear localization signal of SV40 (simian
                     virus40) large T antigen"
                     /translation="PKKKRKV"
     CDS             7110..7175
                     /label="3xFLAG"
                     /product="three tandem FLAG(R) epitope tags, followed by
                     anenterokinase cleavage site"
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             7194..7250
                     /label="P2A"
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             3777..7022
                     /label="NmeN1029A_Cas9"
                     /gene="cas9"
                     /product="NmeN1029A_Cas9 variant"
                     /note="full ORF CDS for engineered NmeN1029A_Cas9 variant"
                     /translation="MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRV
                     FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIK
                     SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA
                     GNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEF
                     GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT
                     KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD
                     NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL
                     KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT
                     EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK
                     EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
                     RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF
                     KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR
                     VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF
                     DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE
                     KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD
                     LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK
                     TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ
                     LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGAINIRIHDLDHKIGKNGILE
                     GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
ORIGIN
        1 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
       61 taaagcaagt aaaacctcta caaatgtggt attggcccat ctctatcggt atcgcgatct
      121 ctcgatttcg atcaagacat tcctttaatg gtcttttctg gacaccacta ggggtcagaa
      181 gtagttcatc aaactttctt ccctccctaa tctcattggt taccttgggc tatcgaaact
      241 taattaacca gtcaagtcag ctacttggcg agatcgactt gtctgggttt cgactacgct
      301 cagaattgcg tcagtcaagt tcgatctggt ccttgctatt gcacccgttc tccgattacg
      361 agtttcattt aaatcatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
      421 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
      481 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
      541 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
      601 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
      661 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
      721 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
      781 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
      841 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
      901 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
      961 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     1021 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     1081 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     1141 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     1201 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgca
     1261 tttaaatttc cgaactctcc aaggccctcg tcggaaaatc ttcaaacctt tcgtccgatc
     1321 catcttgcag gctacctctc gaacgaacta tcgcaagtct cttggccggc cttgcgcctt
     1381 ggctattgct tggcagcgcc tatcgccagg tattactcca atcccgaata tccgagatcg
     1441 ggatcacccg agagaagttc aacctacatc ctcaatcccg atctatccga gatccgagga
     1501 atatcgaaat cggggcgcgc ctggtgtacc gagaacgatc ctctcagtgc gagtctcgac
     1561 gatccatatc gttgcttggc agtcagccag tcggaatcca gcttgggacc caggaagtcc
     1621 aatcgtcaga tattgtactc aagcctggtc acggcagcgt accgatctgt ttaaacctag
     1681 atattgatag tctgatcggt caacgtataa tcgagtccta gcttttgcaa acatctatca
     1741 agagacagga tcagcaggag gctttcgcat gagtattcaa catttccgtg tcgcccttat
     1801 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     1861 aaaagatgct gaagatcagt tgggtgcgcg agtgggttac atcgaactgg atctcaacag
     1921 cggtaagatc cttgagagtt ttcgccccga agaacgcttt ccaatgatga gcacttttaa
     1981 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     2041 ccgcatacac tattctcaga atgacttggt tgagtattca ccagtcacag aaaagcatct
     2101 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     2161 tgcggccaac ttacttctga caacgattgg aggaccgaag gagctaaccg cttttttgca
     2221 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     2281 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacct tgcgtaaact
     2341 attaactggc gaactactta ctctagcttc ccggcaacag ttgatagact ggatggaggc
     2401 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     2461 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     2521 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     2581 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac cgattctagg
     2641 tgcattggcg cagaaaaaaa tgcctgatgc gacgctgcgc gtcttatact cccacatatg
     2701 ccagattcag caacggatac ggcttcccca acttgcccac ttccatacgt gtcctcctta
     2761 ccagaaattt atccttaaga tcccgaatcg tttaaactcg actctggctc tatcgaatct
     2821 ccgtcgtttc gagcttacgc gaacagccgt ggcgctcatt tgctcgtcgg gcatcgaatc
     2881 tcgtcagcta tcgtcagctt acctttttgg cagcgatcgc ggctcccgac atcttggacc
     2941 attagctcca caggtatctt cttccctcta gtggtcataa cagcagcttc agctacctct
     3001 caattcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc tatcaatcgt
     3061 tgcgttacac acacaaaaaa ccaacacaca tccatcttcg atggatagcg attttattat
     3121 ctaactgctg atcgagtgta gccagatcta gtaatcaatt acggggtcat tagttcatag
     3181 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
     3241 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
     3301 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
     3361 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
     3421 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
     3481 attagtcatc gctattacca tgctgatgcg gttttggcag tacatcaatg ggcgtggata
     3541 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
     3601 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
     3661 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctggtt tagtgaaccg
     3721 tcagatcaga tctttgtcga tcctaccatc cactcgacac acccgccagc ggccgcatgg
     3781 cagcattcaa gccaaactca atcaattaca tcctgggact ggacatcggc atcgcatccg
     3841 tcgggtgggc tatggtcgaa atcgacgagg aggagaaccc catccgcctg atcgatctgg
     3901 gcgtgcgcgt gtttgagagg gcagaggtgc ctaagaccgg cgacagcctg gccatggcac
     3961 ggagactggc acgctccgtg aggcgcctga cccggagaag ggcccacaga ctgctgagga
     4021 cacgccggct gctgaagagg gagggcgtgc tgcaggccgc caacttcgat gagaatggcc
     4081 tgatcaagtc cctgcccaat accccttggc agctgagggc agccgccctg gaccgcaagc
     4141 tgacacctct ggagtggtcc gccgtgctgc tgcacctgat caagcaccgg ggctacctgt
     4201 ctcagagaaa gaacgagggc gagacagccg ataaggagct gggcgccctg ctgaagggag
     4261 tggcaggaaa tgcacacgcc ctgcagaccg gcgactttcg cacaccagcc gagctggccc
     4321 tgaacaagtt cgagaaggag agcggccaca tccgcaatca gcggtctgac tatagccaca
     4381 ccttctcccg gaaggatctg caggccgagc tgatcctgct gtttgagaag cagaaggagt
     4441 tcggcaaccc acacgtgtct ggcggcctga aggagggcat cgagacactg ctgatgacac
     4501 agcggcccgc cctgagcggc gacgcagtgc agaagatgct gggacactgc acctttgagc
     4561 cagccgagcc caaggccgcc aagaatacct acacagccga gcggttcatc tggctgacaa
     4621 agctgaacaa tctgaggatc ctggagcagg gaagcgagcg cccactgacc gacacagaga
     4681 gggccaccct gatggatgag ccctaccgca agtccaagct gacatatgca caggcaagga
     4741 agctgctggg cctggaggac accgccttct ttaagggcct gagatacggc aaggataacg
     4801 ccgaggcctc tacactgatg gagatgaagg cctatcacgc catcagcagg gccctggaga
     4861 aggagggcct gaaggacaag aagtccccac tgaatctgtc tcccgagctg caggatgaga
     4921 tcggcaccgc ctttagcctg ttcaagaccg acgaggatat cacaggcaga ctgaaggaca
     4981 ggatccagcc agagatcctg gaggccctgc tgaagcacat cagctttgat aagttcgtgc
     5041 agatcagcct gaaggccctg cggaggatcg tgccactgat ggagcagggc aagaggtacg
     5101 acgaggcctg cgccgaaatc tacggcgatc actatggcaa gaagaacaca gaggagaaaa
     5161 tctacctgcc ccctatcccc gccgatgaga tcaggaaccc tgtggtgctg cgcgccctgt
     5221 ctcaggcaag aaaagtgatc aacggagtgg tgcgccggta cggcagcccc gccagaatcc
     5281 acatcgagac agccagggaa gtgggcaagt cctttaagga cagaaaggag atcgagaaga
     5341 ggcaggagga gaacagaaag gatagggaga aggccgccgc caagttcaga gagtactttc
     5401 ctaatttcgt gggcgagcca aagtccaagg atatcctgaa gctgaggctg tacgagcagc
     5461 agcacggcaa gtgtctgtat tctggcaagg agatcaacct gggccgcctg aatgagaagg
     5521 gctatgtgga gatcgaccac gccctgcctt tttctcggac ctgggacgat agcttcaaca
     5581 ataaggtgct ggtgctgggc tctgagaacc agaataaggg caaccagaca ccctacgagt
     5641 atttcaacgg caaggacaat agccgcgagt ggcaggagtt taaggcaagg gtggagacaa
     5701 gcaggttccc tcggtccaag aagcagagaa tcctgctgca gaagtttgac gaggatggct
     5761 tcaaggagag gaacctgaat gacacccgct acgtgaatcg gtttctgtgc cagttcgtgg
     5821 ccgatagaat gaggctgacc ggcaagggca agaagagagt gtttgcctcc aacggccaga
     5881 tcacaaatct gctgaggggc ttctggggcc tgagaaaggt gagggcagag aacgacaggc
     5941 accacgcact ggatgcagtg gtggtggcat gttctaccgt ggccatgcag cagaagatca
     6001 cacgctttgt gcggtataag gagatgaatg ccttcgacgg caagaccatc gataaggaga
     6061 caggcgaggt gctgcaccag aagacacact ttcctcagcc atgggagttc tttgcccagg
     6121 aagtgatgat ccgggtgttt ggcaagcctg acggcaagcc agagttcgag gaggccgata
     6181 ccctggagaa gctgagaaca ctgctggcag agaagctgag ctccaggccc gaggcagtgc
     6241 acgagtacgt gaccccactg ttcgtgtcta gagcccccaa caggaagatg agcggccagg
     6301 gccacatgga gacagtgaag tccgccaaga gactggacga gggcgtgtct gtgctgaggg
     6361 tgcctctgac acagctgaag ctgaaggatc tggagaagat ggtgaatcgc gagcgggagc
     6421 caaagctgta tgaggccctg aaggcaaggc tggaggcaca caaggacgat cctgccaagg
     6481 cctttgccga gccattctac aagtatgata aggccggcaa cagaacccaa caggtgaagg
     6541 ccgtgagggt ggaacaggtg cagaagacag gcgtgtgggt ccgcaaccac aatggcatcg
     6601 ccgacaatgc taccatggtg cgggtggacg tgtttgagaa gggcgataag tactatctgg
     6661 tgcccatcta cagctggcaa gtggccaagg gcatcctgcc tgatagagcc gtggtgcagg
     6721 gcaaggacga ggaggattgg cagctgatcg acgattcctt caactttaag ttctctctgc
     6781 accccaatga cctggtggaa gtgatcacca agaaggccag gatgtttggc tacttcgcct
     6841 cctgccaccg cggcacaggc gccatcaata tccggatcca cgacctggat cacaagatcg
     6901 gcaagaacgg catcctggag ggcatcggcg tgaagacagc cctgtccttc cagaagtatc
     6961 agatcgacga gctgggcaag gagatcagac cttgtaggct gaagaagcgc ccacccgtgc
     7021 ggggatccgg cggaggcgga agcgggaaaa gaaccgccga cggcagcgaa ttcgagccca
     7081 agaagaagag gaaagtctcg agcggaggcg actacaaaga ccatgacggt gattataaag
     7141 atcatgacat cgattacaag gatgacgatg acaagggagg cggtggaagc ggcgcaacaa
     7201 acttctctct gctgaaacaa gccggagatg tcgaagagaa tcctggaccg atggtgagca
     7261 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     7321 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     7381 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     7441 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     7501 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     7561 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     7621 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     7681 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     7741 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     7801 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     7861 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     7921 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga ccggtcatca
     7981 tcaccatcac catggatccg caggcctctg ctagcttgac tgactgagat acagcgtacc
     8041 ttcagctcac agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc
     8101 agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta
     8161 taagctgcaa taaacaagtt
//
