LOCUS       sequence-227370-        4705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A sgRNA expression plasmid for genome editing in Klebsiella 
            Pneumoniae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4705)
  AUTHORS   Wang Y, Wang S, Chen W, Song L, Zhang Y, Shen Z, Yu F, Li M, Ji Q
  TITLE     Precise and efficient genome editing in Klebsiella pneumoniae using 
            CRISPR-Cas9 and CRISPR-assisted cytidine deaminase.
  JOURNAL   Appl Environ Microbiol. 2018 Sep 14. pii: AEM.01834-18. doi: 
            10.1128/AEM.01834-18.
  PUBMED    30217854
REFERENCE   2  (bases 1 to 4705)
  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: "Appl 
            Environ Microbiol. 2018 Sep 14. pii: AEM.01834-18. doi: 
            10.1128/AEM.01834-18."
COMMENT     Sequence Label: pSGKP-spe
FEATURES             Location/Qualifiers
     source          1..4705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(88..105)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(259..847)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(339..358)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1018..1809)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(1810..1914)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1982..2000
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2038..2060)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2373..2395
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2387..2404
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2388..2404
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2441..2457
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2442..2458
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(join(2520..3854,3855..3941))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        4327..4361
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        4379..4454
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(4487..4503)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4487..4503)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4562..4578)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4562..4578)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4575..4597)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4586..4602
                     /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(4610..4616,4617..4634,4635..4640))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4655..4676
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
       61 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
      121 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
      181 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
      241 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
      301 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      361 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      421 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
      481 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
      541 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      601 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
      661 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
      721 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
      781 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
      841 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
      901 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
      961 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     1021 tttgccgact accttggtga tctcgccttt cacgtagtgg acaaattctt ccaactgatc
     1081 tgcgcgcgag gccaagcgat cttcttcttg tccaagataa gcctgtctag cttcaagtat
     1141 gacgggctga tactgggccg gcaggcgctc cattgcccag tcggcagcga catccttcgg
     1201 cgcgattttg ccggttactg cgctgtacca aatgcgggac aacgtaagca ctacatttcg
     1261 ctcatcgcca gcccagtcgg gcggcgagtt ccatagcgtt aaggtttcat ttagcgcctc
     1321 aaatagatcc tgttcaggaa ccggatcaaa gagttcctcc gccgctggac ctaccaaggc
     1381 aacgctatgt tctcttgctt ttgtcagcaa gatagccaga tcaatgtcga tcgtggctgg
     1441 ctcgaagata cctgcaagaa tgtcattgcg ctgccattct ccaaattgca gttcgcgctt
     1501 agctggataa cgccacggaa tgatgtcgtc gtgcacaaca atggtgactt ctacagcgcg
     1561 gagaatctcg ctctctccag gggaagccga agtttccaaa aggtcgttga tcaaagctcg
     1621 ccgcgttgtt tcatcaagcc ttacggtcac cgtaaccagc aaatcaatat cactgtgtgg
     1681 cttcaggccg ccatccactg cggagccgta caaatgtacg gccagcaacg tcggttcgag
     1741 atggcgctcg atgacgccaa ctacctctga tagttgagtc gatacttcgg cgatcaccgc
     1801 ttcccccata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     1861 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     1921 tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa
     1981 aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct
     2041 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     2101 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc
     2161 ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg
     2221 cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga
     2281 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     2341 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc
     2401 cagtgaattg acgcgtattg ggatggtacc gggccccccc tcgaggtcga cggtatcgat
     2461 acggcatcag agcagattgt actgagagtg caccataatc ggcattttct tttgcgtttt
     2521 tatttgttaa ctgttaattg tccttgttca aggatgctgt ctttgacaac agatgttttc
     2581 ttgcctttga tgttcagcag gaagctaggc gcaaacgttg attgtttgtc tgcgtagaat
     2641 cctctgtttg tcatatagct tgtaatcacg acattgtttc ctttcgcttg aggtacagcg
     2701 aagtgtgagt aagtaaaggt tacatcgtta ggatcaagat ccatttttaa cacaaggcca
     2761 gttttgttca gcggcttgta tgggccagtt aaagaattag aaacataacc aagcatgtaa
     2821 atatcgttag acgtaatgcc gtcaatcgtc atttttgatc cgcgggagtc agtgaacaga
     2881 taccatttgc cgttcatttt aaagacgttc gcgcgttcaa tttcatctgt tactgtgtta
     2941 gatgcaatca gcggtttcat cacttttttc agtgtgtaat catcgtttag ctcaatcata
     3001 ccgagagcgc cgtttgctaa ctcagccgtg cgttttttat cgctttgcag aagtttttga
     3061 ctttcttgac ggaagaatga tgtgcttttg ccatagtatg ctttgttaaa taaagattct
     3121 tcgccttggt agccatcttc agttccagtg tttgcttcaa atactaagta tttgtggcct
     3181 ttatcttcta cgtagtgagg atctctcagc gtatggttgt cgcctgagct gtagttgcct
     3241 tcatcgatga actgctgtac attttgatac gtttttccgt caccgtcaaa gattgattta
     3301 taatcctcta caccgttgat gttcaaagag ctgtctgatg ctgatacgtt aacttgtgca
     3361 gttgtcagtg tttgtttgcc gtaatgttta ccggagaaat cagtgtagaa taaacggatt
     3421 tttccgtcag atgtaaatgt ggctgaacct gaccattctt gtgtttggtc ttttaggata
     3481 gaatcatttg catcgaattt gtcgctgtct ttaaagacgc ggccagcgtt tttccagctg
     3541 tcaatagaag tttcgccgac tttttgatag aacatgtaaa tcgatgtgtc atccgcattt
     3601 ttaggatctc cggctaatgc aaagacgatg tggtagccgt gatagtttgc gacagtgccg
     3661 tcagcgtttt gtaatggcca gctgtcccaa acgtccaggc cttttgcaga agagatattt
     3721 ttaattgtgg acgaatcgaa ctcaggaact tgatattttt catttttttg ctgttcaggg
     3781 atttgcagca tatcatggcg tgtaatatgg gaaatgccgt atgtttcctt atatggcttt
     3841 tggttcgttt ctttcgcaaa cgcttgagtt gcgcctcctg ccagcagtgc ggtagtaaag
     3901 gttaatactg ttgcttgttt tgcaaacttt ttgatgttca tcgttcatgt ctcctttttt
     3961 atgtactgtg ttagcggtct gcttcttcca gccctcctgt ttgaagatgg caagttagtt
     4021 acgcacaata aaaaaagacc taaaatatgt aaggggtgac gccaaagtat acactttgcc
     4081 ctttacacat tttaggtctt gcctgcttta tcagtaacaa acccgcgcga tttacttttc
     4141 gacctcattc tattagactc tcgtttggat tgcaactggt ctattttcct cttttgtttg
     4201 atagaaaatc ataaaaggat ttgcagacta cgggcctaaa gaactaaaaa atctatctgt
     4261 ttcttttcat tctctgtatt ttttatagtt tctgttgcat gggcataaag ttgcaagctt
     4321 gatatcttga cagctagctc agtcctaggt ataatactag tcgagaccat tggtctcagt
     4381 tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg
     4441 caccgagtcg gtgctttttt tgatatcgaa ttcctgcagc ccgggggatc cactagttct
     4501 agagcggccg ccaccgcggt ggagctcatc ccaatggcgc gccgagcttg gctcgagcat
     4561 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4621 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4681 cgttgcgctc actgcccgct ttcca
//
