LOCUS       Exported                4729 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  A sgRNA expression plasmid for genome editing in Klebsiella 
            Pneumoniae.
ACCESSION   .
VERSION     .
KEYWORDS    pSGKP-km
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4729)
  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 4729)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl 
            Environ Microbiol. 2018 Sep 14. pii: AEM.01834-18. doi: 
            10.1128/AEM.01834-18."
FEATURES             Location/Qualifiers
     source          1..4729
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        97..131
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        149..224
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(257..273)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(257..273)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(332..348)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(332..348)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(345..367)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    356..372
                     /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(380..410)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    425..446
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(563..580)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(734..1322)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(814..833)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1493..2308)
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2216..2235
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(2309..2413)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2481..2499
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2537..2559)
                     /label=pGEX 3
                     /note="pGEX vectors, reverse primer"
     primer_bind     2872..2894
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2886..2903
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2887..2903
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2940..2956
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2941..2957
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3019..4440)
                     /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"