LOCUS       sequence-330962-        7687 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Vector for expression of the SpCas9 KG variant with sgRNA in E. 
            coli: KG(SpCas9, D1332K/R1333G) and UV5-sgRNA (hEGFP spacer).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7687)
  AUTHORS   Goldberg GW, Spencer JM, Giganti DO, Camellato BR, Agmon N, Ichikawa
            DM, Boeke JD, Noyes MB
  TITLE     Engineered dual selection for directed evolution of SpCas9 PAM 
            specificity.
  JOURNAL   Nat Commun. 2021 Jan 13;12(1):349. doi: 10.1038/s41467-020-20650-x.
  PUBMED    33441553
REFERENCE   2  (bases 1 to 7687)
  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 
            Commun."; date: "2021-01-13"; volume: "12(1):349. doi"; pages: " 
            10.1038/s41467-020-20650-x"
COMMENT     Sequence Label: pGG422_UV5
FEATURES             Location/Qualifiers
     source          1..7687
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..24,25..31)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    39..55
                     /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     44..66
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     CDS             294..4400
                     /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIKGKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     misc_RNA        4496..4571
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(4583..4607)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     rep_origin      4737..5192
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4824..4843)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5034..5055
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5218..5322
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5323..5391,5392..6183)
                     /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(5541..5560)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6354..6942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6843..6862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7096..7113
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7128..7268
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7194..7213)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7354..7376
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7370..7561)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tttacacttt atgcttccgg ctcgtataat gtgtcgactt gtgagcggat aacaatttca
       61 cacaggaaac agctatgcct caacagcagc aaatgcaacc tcccaattca agtgcggaca
      121 acaacccttt gcaacagcaa tcatcacaaa ataccgtacc aaacgtcctc aaccaaatta
      181 accaaatctt ttctccagag gagcaacgca gcttattaca agaagccatc gaaacctgca
      241 agaattttga aaaaacacaa ttgtaagtct cggctccaag aaggagatat accatggata
      301 agaaatactc aataggctta gatatcggca caaatagcgt cggatgggcg gtgatcactg
      361 atgaatataa ggttccgtct aaaaagttca aggttctggg aaatacagac cgccacagta
      421 tcaaaaaaaa tcttataggg gctcttttat ttgacagtgg agagacagcg gaagcgactc
      481 gtctcaaacg gacagctcgt agaaggtata cacgtcggaa gaatcgtatt tgttatctac
      541 aggagatttt ttcaaatgag atggcgaaag tagatgatag tttctttcat cgacttgaag
      601 agtctttttt ggtggaagaa gacaagaagc atgaacgtca tcctattttt ggaaatatag
      661 tagatgaagt tgcttatcat gagaaatatc caactatcta tcatctgcga aaaaaattgg
      721 tagattctac tgataaagcg gatttgcgct taatctattt ggccttagcg catatgatta
      781 agtttcgtgg tcattttttg attgagggag atttaaatcc tgataatagt gatgtggaca
      841 aactatttat ccagttggta caaacctaca atcaattatt tgaagaaaac cctattaacg
      901 caagtggagt agatgctaaa gcgattcttt ctgcacgatt gagtaaatca agacgattag
      961 aaaatctcat tgctcagctc cccggtgaga agaaaaatgg cttatttggg aatctcattg
     1021 ctttgtcatt gggtttgacc cctaatttta aatcaaattt tgatttggca gaagatgcta
     1081 aattacagct ttcaaaagat acttacgatg atgatttaga taatttattg gcgcaaattg
     1141 gagatcaata tgctgatttg tttttggcag ctaagaattt atcagatgct attttacttt
     1201 cagatatcct aagagtaaat actgaaataa ctaaggctcc cctatcagct tcaatgatta
     1261 aacgctacga tgaacatcat caagacttga ctcttttaaa agctttagtt cgacaacaac
     1321 ttccagaaaa gtataaagaa atcttttttg atcaatcaaa aaacggatat gcaggttata
     1381 ttgatggggg agcgagccaa gaagaatttt ataaatttat caaaccaatt ttagaaaaaa
     1441 tggatggtac tgaggaatta ttggtgaaac taaatcgtga agatttgctg cgcaagcaac
     1501 ggacctttga caacggctct attccccatc aaattcactt gggtgagctg cacgctattt
     1561 tgagaagaca agaagacttt tatccatttt taaaagacaa tcgtgagaag attgaaaaaa
     1621 tcttgacttt tcgcatccct tattatgttg gtccattggc gcgtggcaat agtcgttttg
     1681 catggatgac tcggaagtct gaagaaacaa ttaccccgtg gaattttgaa gaagttgtcg
     1741 ataaaggtgc ttcagctcaa tcatttattg aacgcatgac aaactttgat aaaaatcttc
     1801 caaatgaaaa agtactacca aaacatagtt tgctttatga gtattttacg gtttataacg
     1861 aattgacaaa ggtcaaatat gttactgaag gaatgcgaaa accagcattt ctttcaggtg
     1921 aacagaagaa agccattgtt gatttactct tcaaaacaaa tcgaaaagta accgttaagc
     1981 aattaaaaga agattatttc aaaaaaatag aatgttttga tagtgttgaa atttcaggag
     2041 ttgaagatag atttaatgct tcattaggga cataccatga tttgctaaaa attattaaag
     2101 ataaagattt tttggataat gaagaaaatg aagatatctt agaggatatt gttttaacat
     2161 tgaccttatt tgaagatagg gagatgattg aggaaagact taaaacatat gctcacctct
     2221 ttgatgataa ggtgatgaaa cagcttaaac gtcgccgtta tactggttgg ggacgtttgt
     2281 ctcgaaaatt gattaatggt attagggata agcaatctgg caaaacaata ttagattttt
     2341 tgaaatcaga tggttttgcc aatcgcaatt ttatgcagct gatccatgat gatagtttga
     2401 catttaaaga agacattcaa aaagcacaag tgtctggaca aggcgatagt ttacatgaac
     2461 atattgcaaa tttagctggt agccctgcta ttaaaaaagg tattttacag actgtaaaag
     2521 ttgttgatga attggtcaaa gtaatggggc ggcataagcc agaaaatatc gttattgaaa
     2581 tggcacgtga aaatcagaca actcaaaagg gccagaaaaa ttcgcgagag cgtatgaaac
     2641 gaatcgaaga aggtatcaaa gaattaggaa gtcagattct taaagagcat cctgttgaaa
     2701 atactcaatt gcaaaatgaa aagctctatc tctattatct ccaaaatgga agagacatgt
     2761 atgtggacca agaattagat attaatcgtt taagtgatta tgatgtcgat cacattgttc
     2821 cacaaagttt ccttaaagac gattcaatag acaataaggt cttaacgcgt tctgataaaa
     2881 atcgtggtaa atcggataac gttccaagtg aagaagtagt caaaaagatg aaaaactatt
     2941 ggagacaact tctaaacgcc aagttaatca ctcaacgtaa gtttgataat ttaacgaaag
     3001 ctgaacgtgg aggtttgagt gaacttgata aagctggttt tatcaaacgc caattggttg
     3061 aaactcgcca aatcactaag catgtggcac aaattttgga tagtcgcatg aatactaaat
     3121 acgatgaaaa tgataaactt attcgagagg ttaaagtgat taccttaaaa tctaaattag
     3181 tttctgactt ccgaaaagat ttccaattct ataaagtacg tgagattaac aattaccatc
     3241 atgcccatga tgcgtatcta aatgccgtcg ttggaactgc tttgattaag aaatatccaa
     3301 aacttgaatc ggagtttgtc tatggtgatt ataaagttta tgatgttcgt aaaatgattg
     3361 ctaagtctga gcaagaaata ggcaaagcaa ccgcaaaata tttcttttac tctaatatca
     3421 tgaacttctt caaaacagaa attacacttg caaatggaga gattcgcaag cggccgctaa
     3481 tcgaaactaa tggggaaact ggagaaattg tctgggataa agggcgagat tttgccacag
     3541 tgcgcaaagt attgtccatg ccccaagtca atattgtcaa gaaaacagaa gtacagaccg
     3601 gtggattctc caaggagtca attttaccaa aaagaaattc ggacaagctt attgctcgta
     3661 aaaaagactg ggacccaaaa aaatatggtg gttttgatag tccaacggta gcttattcag
     3721 tcctagtggt tgctaaggtg gaaaaaggga aatcgaagaa gttaaaatcc gttaaagagt
     3781 tactagggat cacaattatg gaaagaagtt cctttgaaaa aaatccgatt gactttttag
     3841 aagctaaagg atataaggaa gttaaaaaag acttaatcat taaactacct aaatatagtc
     3901 tttttgagtt agaaaacggt cgtaaacgga tgctggctag tgccggagaa ttacaaaaag
     3961 gaaatgagct ggctctgcca agcaaatatg tgaatttttt atatttagct agtcattatg
     4021 aaaagttgaa gggtagtcca gaagataacg aacaaaaaca attgtttgtg gagcagcata
     4081 agcattattt agatgagatt attgagcaaa tcagtgaatt ttctaagcgt gttattttag
     4141 cagatgccaa tttagataaa gttcttagtg catataacaa acatagagac aaaccaatac
     4201 gtgaacaagc agaaaatatt attcatttat ttacgttgac gaatcttgga gcgcccgctg
     4261 cgttcaagta cttcgacacc acgatcaagg ggaagaggta cacgtctaca aaagaagttc
     4321 tagatgccac tcttatccat caatccatca ctggtcttta tgaaacacgc attgatttga
     4381 gtcagctagg aggtgactga tagggatccg gtacctaata gtgagtcgag ctgcccgggt
     4441 tgacacgcaa gctcagtcct aggtataata ctagtcccat cctggtcgag ctggagtttt
     4501 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
     4561 cgagtcggtg ctttttttct acacccagct ttcttgtaca aagttggcat taactcgagg
     4621 ctagagacta gaaaaaggcc gacaagtccc gctccgctga agatcctggc gtaatagcga
     4681 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc
     4741 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4801 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4861 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4921 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4981 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     5041 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     5101 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     5161 gaattttaac aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc
     5221 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     5281 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     5341 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     5401 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     5461 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     5521 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     5581 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     5641 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     5701 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     5761 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     5821 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     5881 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     5941 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     6001 tggtttattg ctgataaatc tggagccggt gagcgtggtt ctcgcggtat cattgcagca
     6061 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     6121 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     6181 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     6241 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     6301 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     6361 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     6421 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     6481 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     6541 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     6601 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     6661 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     6721 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     6781 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     6841 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     6901 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     6961 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     7021 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     7081 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     7141 tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc
     7201 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     7261 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     7321 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     7381 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     7441 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     7501 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     7561 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     7621 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgggaat
     7681 tccgggc
//
