LOCUS       sequence-431527-        5762 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (Cas protein) NGRR(T) PAM.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5762)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 5762)
  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: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA180
FEATURES             Location/Qualifiers
     source          1..5762
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             445..3600
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     primer_bind     complement(3672..3688)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3672..3688)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3685..3707)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3696..3712
                     /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)."
     promoter        complement(join(3720..3726,3727..3744,3745..3750))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3765..3786
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3903..3920)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4074..4662)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4154..4173)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4840..5649)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5563..5582
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5641..5660
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagactgg cagcaagcgc aactacatcc tgggcctgga cattggcatc
      481 acctcagttg gctatggcat catcgactac gagacccggg acgtcatcga cgctggcgtc
      541 cgcctcttca aggaggccaa cgtcgagaac aacgagggcc ggcggtccaa gcgcggggcc
      601 cgccggctca agcgccgccg ccgccaccgc atccagcgcg tgaaaaagtt gctcttcgac
      661 tacaacttgc tcaccgacca ctccgagctg tctggcatca acccgtacga ggcccgggtc
      721 aagggcctct cgcaaaagtt gtccgaggag gagttctctg ctgccctgct gcacctggcc
      781 aagcgccgcg gggtccacaa cgtcaacgaa gttgaggagg acaccggcaa cgaactttcc
      841 accaaggagc agatctcccg caactccaag gccctggagg agaagtacgt cgcggagctc
      901 caacttgagc gcctcaagaa ggacggcgag gtgcgcggct ccatcaaccg cttcaagacc
      961 tccgactacg tcaaggaggc caagcagctg ctcaaggtcc agaaggccta ccaccaactt
     1021 gaccagtcct tcatcgacac ctacatcgac ctgctggaga cccgccgcac ctactacgag
     1081 gggcctggcg agggctctcc cttcggctgg aaggacatca aggagtggta cgagatgctc
     1141 atgggccact gcacctactt cccggaggag ctgcgctcgg tcaagtacgc ctacaacgcc
     1201 gacctctaca acgcgctcaa cgacctcaac aacttggtca tcacccgcga cgagaacgaa
     1261 aagttggagt actacgaaaa gttccagatc atcgaaaacg tgttcaagca gaagaagaag
     1321 ccaactttga agcagatcgc caaggagatc ctggtcaacg aggaggacat caagggctac
     1381 cgggtcacct ccaccggcaa gcccgagttc accaacttaa aggtctacca tgacatcaag
     1441 gacatcaccg cccgcaagga gatcatcgag aacgcggagc tgctggacca gatcgccaag
     1501 atcctcacca tctaccaaag ttccgaggac atccaggagg agctgaccaa cttaaactcg
     1561 gagcttaccc aggaggagat cgagcagatc tccaacttaa agggctacac tggcacccac
     1621 aacttgtccc tcaaggccat caacttgatc ctggacgaac tttggcatac caacgacaac
     1681 cagatcgcca tcttcaaccg actaaagttg gtgcccaaga aagttgacct gtcccaacag
     1741 aaggagatcc caactaccct ggtcgacgac ttcatcctct cgcccgtggt caagcgcagc
     1801 ttcatccagt ccatcaaggt catcaacgcc atcatcaaga agtacggcct gcccaacgac
     1861 atcatcatcg agttggcacg cgagaagaac tccaaggatg cccagaagat gatcaacgag
     1921 atgcagaagc gcaaccgcca gaccaacgag cgcatcgagg agatcatccg caccaccggc
     1981 aaggagaacg ccaagtacct gatcgagaag ataaagttgc atgacatgca ggagggcaag
     2041 tgcctctact ccctggaggc catccccctc gaggacctgc tcaacaaccc cttcaactac
     2101 gaagttgacc acatcatccc gcgctcggtg tccttcgaca acagcttcaa caacaaggtc
     2161 ctggtcaagc aggaggagaa ctccaagaag ggcaaccgga cccccttcca gtacctgtcc
     2221 tcctcggact ccaagatctc ctacgaaact ttcaagaagc acatcctcaa cttggcaaag
     2281 ggcaagggcc gcatctccaa gaccaagaag gagtacctgc tggaggagcg cgacatcaac
     2341 cgcttctcgg tgcagaagga cttcatcaac cgcaacctgg tggacacccg ctacgcaacc
     2401 cgtggcctca tgaacctgct gcgcagctac ttccgggtca acaacttgga cgtcaaagtt
     2461 aagtcgatca acggtggctt caccagcttc ctgcgccgca agtggaagtt caagaaggag
     2521 cgcaacaagg gctacaagca ccatgcggag gacgccctca tcatcgccaa cgccgacttc
     2581 atcttcaagg agtggaaaaa gttggacaag gccaagaagg tcatggagaa ccagatgttc
     2641 gaggagaagc aggccgagtc catgccggag atcgagacag agcaggagta caaggagatc
     2701 ttcatcacgc cgcaccagat caagcacatc aaggacttca aggactacaa gtactcccac
     2761 cgcgtggaca agaagcccaa ccgcgagctc atcaacgaca ccctctactc cacccgcaag
     2821 gacgacaagg gcaacaccct catcgtcaac aacttaaacg gcctctacga caaggacaac
     2881 gacaagttga aaaagttgat caacaagagc cccgaaaagt tgctcatgta ccaccatgac
     2941 ccgcagacgt accaaaagtt gaagttgatc atggagcagt acggtgacga gaagaacccg
     3001 ctctacaagt actacgagga gaccggcaac tacctcacca agtactccaa gaaggacaac
     3061 ggcccggtca tcaagaagat caagtactac ggcaacaagt tgaatgccca cctggacatc
     3121 accgacgact accccaactc ccgcaacaaa gttgtaaagt tgtcgctaaa gccgtaccgc
     3181 ttcgacgtct acctggacaa cggcgtctac aagttcgtga ccgtcaagaa cctggacgtc
     3241 atcaagaagg agaactacta cgaggtcaac tccaagtgct acgaggaggc caaaaagttg
     3301 aagaagatct ccaaccaggc cgagttcatc gccagcttct acaacaacga cctcatcaag
     3361 atcaacggcg agctctaccg cgtcatcggg gtcaacaacg acctgctgaa ccgcatcgag
     3421 gtcaacatga tcgacatcac ctaccgcgag tacctcgaga acatgaacga caagcgcccg
     3481 cctcgcatca tcaagaccat cgcctccaag acccagtcca tcaagaagta ctccaccgac
     3541 atccttggca acttgtacga ggtcaagagc aagaagcacc ctcagatcat caagaagggc
     3601 gggtccaagt cttcatcgga tcccgggccc gtcgactgca gaggcctgca tgcaagcttg
     3661 gtgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     3721 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     3781 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     3841 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     3901 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     3961 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     4021 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     4081 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     4141 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     4201 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     4261 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     4321 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     4381 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     4441 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     4501 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     4561 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     4621 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     4681 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     4741 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaagc
     4801 ccaatctgaa taatgttaca accaattaac caattctgat tagaaaaact catcgagcat
     4861 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     4921 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     4981 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     5041 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     5101 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     5161 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     5221 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     5281 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     5341 tgtttttccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     5401 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     5461 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     5521 cccatacaag cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     5581 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctcgacgttt cccgttgaat
     5641 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     5701 tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacgg gccagagctg
     5761 ca
//
