LOCUS       sequence-431396-        6707 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (Cas protein) nickase Cas for prime editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6707)
  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 6707)
  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: AA060
FEATURES             Location/Qualifiers
     source          1..6707
                     /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             join(445..2958,2959..2961,2962..4545)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             445..4545
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     complement(4617..4633)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4617..4633)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4630..4652)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4641..4657
                     /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(4665..4671,4672..4689,4690..4695))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4710..4731
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4848..4865)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5019..5607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5099..5118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5785..6594)
                     /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     6508..6527
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     6586..6605
                     /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 cagcgacaag aagtacagca tcggcctgga catcggcacc
      481 aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa gaaattcaag
      541 gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc cctgctgttc
      601 gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag aagatacacc
      661 agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat ggccaaggtg
      721 gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga taagaagcac
      781 gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga gaagtacccc
      841 accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga cctgcggctg
      901 atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat cgagggcgac
      961 ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca gacctacaac
     1021 cagctgttcg aggaaaaccc catcaacgct agcggcgtgg acgccaaggc catcctgtct
     1081 gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc cggcgagaag
     1141 aagaatggcc tgttcggcaa cctgattgcc ctgagcctgg gcctgacccc caacttcaag
     1201 agcaacttcg acctggccga ggatgccaaa ctgcagctga gcaaggacac ctacgacgac
     1261 gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt tctggccgcc
     1321 aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac cgagatcacc
     1381 aaggcccccc tgagcgcctc tatgatcaag agatacgacg agcaccacca ggacctgacc
     1441 ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat tttcttcgac
     1501 cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga agagttctac
     1561 aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct cgtgaagctg
     1621 aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcagcat cccccaccag
     1681 atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta cccattcctg
     1741 aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta ctacgtgggc
     1801 cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga ggaaaccatc
     1861 accccctgga acttcgagga agtggtggac aagggcgctt ccgcccagag cttcatcgag
     1921 cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa gcacagcctg
     1981 ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt gaccgaggga
     2041 atgagaaagc ccgccttcct gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc
     2101 aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag
     2161 tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca
     2221 taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga ggaaaacgag
     2281 gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga gatgatcgag
     2341 gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca gctgaagcgg
     2401 cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat ccgggacaag
     2461 cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa cagaaacttc
     2521 atgcagctga tccacgacga cagcctgacc tttaaagagg acatccagaa agcccaggtg
     2581 tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag ccccgccatt
     2641 aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt gatgggccgg
     2701 cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac ccagaaggga
     2761 cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga gctgggcagc
     2821 cagatcctga aagaacaccc agtggaaaac acccagctgc agaacgagaa gctgtacctg
     2881 tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat caaccggctg
     2941 tccgactacg atgtggacgc tatcgtgcct cagagctttc tgaaggacga ctccatcgac
     3001 aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt gccctccgaa
     3061 gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa gctgattacc
     3121 cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga actggataag
     3181 gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca cgtggcacag
     3241 atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat ccgggaagtg
     3301 aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt ccagttttac
     3361 aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa cgccgtcgtg
     3421 ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta cggcgactac
     3481 aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg caaggctacc
     3541 gccaagtact tcttctacag caacatcatg aactttttca agaccgagat taccctggcc
     3601 aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg ggagatcgtg
     3661 tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc ccaagtgaat
     3721 atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat cctgcccaag
     3781 aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa gtacggcggc
     3841 ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga aaagggcaag
     3901 tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga aagaagcagc
     3961 ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt gaaaaaggac
     4021 ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg gaagagaatg
     4081 ctggcctctg ccggcgaact gcagaaggga aacgaactgg ccctgccctc caaatatgtg
     4141 aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga ggataatgag
     4201 cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat cgagcagatc
     4261 agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt gctgtccgcc
     4321 tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat ccacctgttt
     4381 accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac catcgaccgg
     4441 aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca gagcatcacc
     4501 ggcctgtacg agacacgaat cgatttgtct cagctgggag gcgatgggtc caagtcttca
     4561 tcggatcccg ggcccgtcga ctgcagaggc ctgcatgcaa gcttggtgta atcatggtca
     4621 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4681 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4741 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4801 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4861 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4921 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4981 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5041 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5101 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5161 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5221 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5281 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5341 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5401 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5461 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5521 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5581 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5641 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5701 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caagcccaat ctgaataatg
     5761 ttacaaccaa ttaaccaatt ctgattagaa aaactcatcg agcatcaaat gaaactgcaa
     5821 tttattcata tcaggattat caataccata tttttgaaaa agccgtttct gtaatgaagg
     5881 agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt ctgcgattcc
     5941 gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa ggttatcaag
     6001 tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc
     6061 tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac tcgcatcaac
     6121 caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat cgctgttaaa
     6181 aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac
     6241 aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt ttccggggat
     6301 cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga tggtcggaag
     6361 aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat cattggcaac
     6421 gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat acaagcgata
     6481 gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat ataaatcagc
     6541 atccatgttg gaatttaatc gcggcctcga cgtttcccgt tgaatatggc tcataacacc
     6601 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc
     6661 ttgtgcaatg taacatcaga gattttgaga cacgggccag agctgca
//
