LOCUS       sequence-429368-        7587 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SlugABE-NNG .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7587)
  AUTHORS   Qi T, Wang Y, Yang Y, Gao S, Liu J, Huang Q, Tian Y, Tang J, Zheng 
            WV, Wang Y
  TITLE     Phage-assisted evolution of compact Cas9 variants targeting a simple
            NNG PAM.
  JOURNAL   Nat Chem Biol. 2023 Dec 5. doi: 10.1038/s41589-023-01481-5.
  PUBMED    38052959
REFERENCE   2  (bases 1 to 7587)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2023 Dec 5. doi: 10.1038/s41589-023-01481-5."
COMMENT     Sequence Label: pAAV-SlugABE-NNG
FEATURES             Location/Qualifiers
     source          1..7587
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..356
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             406..426
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4222..4242
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4252..4476
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_RNA        complement(4484..4559)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(4579..4827)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4637..4656)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4807..4827)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4850..4990
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4850..4979
                     /label=AAV2 ITR
     rep_origin      5065..5520
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5152..5171)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5362..5383
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5537..5556)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5656..5678
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5716..5734)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5802..5906
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5907..5975,5976..6767)
                     /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     complement(6125..6144)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6938..7526
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7427..7446
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgggcag agcgcacatc gcccacagtc cccgagaagt
      181 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg
      241 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
      301 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggacc
      361 ggtgccacca tgaaacggac agccgacgga agcgagttcg agtcaccaaa gaagaagcgg
      421 aaagtctctg aggtggagtt ttcccacgag tactggatga gacatgccct gaccctggcc
      481 aagagggcac gggatgagag ggaggtgcct gtgggagccg tgctggtgct gaacaataga
      541 gtgatcggcg agggctggaa cagagccatc ggcctgcacg acccaacagc ccatgccgaa
      601 attatggccc tgagacaggg cggcctggtc atgcagaact acagactgat tgacgccacc
      661 ctgtacgtga cattcgagcc ttgcgtgatg tgcgccggcg ccatgatcca ctctaggatc
      721 ggccgcgtgg tgtttggcgt gaggaactca aaaagaggcg ccgcaggctc cctgatgaac
      781 gtgctgaact accccggcat gaatcaccgc gtcgaaatta ccgagggaat cctggcagat
      841 gaatgtgccg ccctgctgtg cgatttctat cggatgccta gacaggtgtt caatgctcag
      901 aagaaggccc agagctccat caactccgga ggatctagcg gaggctcctc tggctctgag
      961 acacctggca caagcgagag cgcaacacct gaaagcagcg ggggcagcag cggggggtca
     1021 aaccaaaaat tcatactggg actggccatc ggaatcacca gcgtgggcta cggcctgatc
     1081 gactacgaga caaagaatat catcgatgcc ggcgttagac tgttccccga ggccaacgtg
     1141 gaaaacaacg agggaagaag gtccaaacgt ggaagcagaa gactgaagcg acgccgcatt
     1201 cacagacttg aacgggtgaa gaagctgctc gaggattata atctgctgga tcagtcccag
     1261 attcctcagt ctacaaaccc ctacgccatc cgcgtgaagg gcctgtctga agccctgagc
     1321 aaggacgaac tcgtgattgc cctgctccat atcgccaaga gaagaggcat ccacaagatc
     1381 gacgtgatcg acagcaacga cgacgtgggg aacgagctca gcaccaagga acagctgaat
     1441 aagaacagca agctgctgaa agacaaattt gtgtgccaga tccagctgga aagaatgaat
     1501 gagggccagg tgcggggaga gaaaaaccgg ttcaagaccg ctgatatcat caaggaaatc
     1561 atccagctgc tgaatgtgca gaagaacttc caccagctgg acgagaactt catcaacaag
     1621 tacatcgaac tggttgagat gagacgggaa tacttcgagg gccccggcaa gggcagtcca
     1681 tatggctggg aaggcgaccc taaggcttgg tacgagacac tgatgggcca ctgcacctac
     1741 ttcccagatg agctgagaag cgtgaaatac gcctacagtg ccgacctgtt caacgctctg
     1801 aacgacctga acaacctggt catccaaaga gatggactgt ctaagctcga gtatcatgag
     1861 aagtatcaca tcatcgagaa cgtgttcaag cagaagaaga aacctacact gaagcagatc
     1921 gccaatgaga tcaatgtcaa ccctgaagat atcaagggct acagaatcac aaagtctggc
     1981 aagccccagt ttaccgagtt taagctctac cacgacctga aaagcgtgct gtttgaccag
     2041 agcatcctgg agaacgaaga cgtgctggac cagatcgctg agatcctgac catctaccag
     2101 gacaaggata gcatcaaatc taagctgacg gaactggaca tcctgctgaa cgaggaagat
     2161 aaggaaaaca tcgcccagct gactggctac accgggaccc accggctcag cctgaaatgc
     2221 atccggctgg tcctggagga gcagtggtat tctagccgga accagatgga aatcttcacc
     2281 cacctgaaca ttaagcctaa gaagatcaac ctgacagccg ccaacaagat cccgaaggct
     2341 atgatcgacg agttcatcct gagccctgtg gtgaagagga ccttcggcca ggccattaac
     2401 cttattaaca agatcataga aaagtacggc gtgcctgaag atatcatcat cgagctggcc
     2461 agagaaaata atagcaagga caagcagaag ttcatcaatg agatgcagaa aaagaacgag
     2521 aacaccagaa agagaattaa cgaaatcatc ggcaagtatg gcaaccagaa cgccaagaga
     2581 ctggtcgaga agattagact gcacgacgag caggagggca agtgcctgta ctcactggaa
     2641 agcatccctc tggaggacct gctgaacaac cccaaccact acgaggtgga ccacatcatt
     2701 ccaagatctg tgtccttcga caactcttac cacaacaaag tgctcgtgaa gcagagcgag
     2761 aactccaaaa aatccaacct gaccccttac cagtacttta acagcggcaa gtccaagctc
     2821 tcttacaacc agtttaaaca acacatcctg aacctgagca agtcccagga tagaatcagc
     2881 aaaaaaaaga aagagtatct gctggaagaa cgggacatca acaagttcga ggtgcaaaaa
     2941 gagttcatca atagaaacct ggtggatacc cggtacgcca caagagagct gacaaactac
     3001 ctgaaggcct acttcagcgc caacaatatg aacgtgaagg tgaaaacgat caacggcagc
     3061 ttcaccgatt acctgcggaa agtgtggaag tttaagaagg aacggaacca cggctacaag
     3121 caccacgccg aggacgccct gattatcgct aatgccgatt tcctgttcaa agagaacaag
     3181 aagctgaaag ccgtgaactc tgtgctggaa aaacctgaga tcgagagcaa gcagctggat
     3241 atccaggtgg atagcgagga taactacagc gaaatgttca tcatccctaa gcaggtccag
     3301 gacatcaagg acttcagaaa cttcaagtac agccacagag tggacaagaa gcctaacaga
     3361 cggctgatca acgatacact gtacagcacc cggaagaagg acaactccac ctacatcgtg
     3421 cagaccatca aagatatcta tgccaaagat aataccaccc tgaagaagca gtttgacaag
     3481 tcacccgaga agttcctcat gtaccaacac gatccgcgga ccttcgagaa gttggaagtg
     3541 atcatgaagc agtacgctaa tgagaagaat cctctggcca agtaccacga ggaaacaggc
     3601 gagtacctga ccaaatacag caaaaaaaac aacggcccta tcgtgaaaag cctgaagtac
     3661 attggaaaca agctgggcag acacctagat gtgacccacc agttcaagag cagcaccaag
     3721 aagttggtga agcggagcat caagccttat agattcgacg tctacctgac cgacaaggga
     3781 tataagttca tcaccatcag ctacctggac gtgctgaaga aagacaatta ctactacata
     3841 cccgaacaga agtacgacaa gctcaaactg ggcaaggcca tcgacaaaaa cgccaagttt
     3901 atcgctagct tctacaagaa tgatctgatc aagctggacg gcgagatcta caagatcatc
     3961 ggcgtgagta gcgacaccag aaacatgatc gaactggatc tgcctgacat cagatacaaa
     4021 gaatactgcg agctgaacaa tatcaagggc aaacctagaa tcataaagac catcggcaaa
     4081 aaggtgaata gcatcgaaaa actgacaacc gacgtgctgg gcaacgtgtt caccaacacc
     4141 cagtacacaa aacctcagct gctgttcaag cgaggaaatt ctggcggctc aaaaagaacc
     4201 gccgacggca gcgaattcga gcccaagaag aagaggaaag tctaatagcg actgtgcctt
     4261 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     4321 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     4381 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     4441 atagcaggca tgctggggat gcggtgggct ctatggaaaa aaatctcgcc aacaagttga
     4501 cgagataaac acggcatttt gccttgtttt agtagattct gtttccagag tactaaaacc
     4561 gaagagcatc gctcttctgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc
     4621 gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac
     4681 tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt
     4741 gtttacagtc aaattaattc caattatctc tctaacagcc ttgtatcgta tatgcaaata
     4801 tgaaggaatc atgggaaata ggccctccac gtgcggaccg agcggccgca ggaaccccta
     4861 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     4921 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     4981 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     5041 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5101 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     5161 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5221 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5281 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5341 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     5401 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     5461 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     5521 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     5581 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     5641 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     5701 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     5761 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     5821 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5881 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5941 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     6001 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     6061 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     6121 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     6181 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     6241 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     6301 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     6361 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     6421 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     6481 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     6541 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     6601 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     6661 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     6721 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     6781 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6841 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6901 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6961 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     7021 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     7081 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     7141 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     7201 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     7261 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7321 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7381 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7441 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7501 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7561 ccttttgctg gccttttgct cacatgt
//
