LOCUS       40924_39942        4729 bp DNA     circular SYN 13-MAY-2021
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 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)
  TITLE     Direct Submission
REFERENCE   3  (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."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..4729
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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(332..348)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(356..372)
                     /label=lac operator
                     /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    complement(425..446)
                     /label=CAP binding site
                     /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"
     CDS             complement(1496..2308)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(2309..2413)
                     /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     2887..2903
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2941..2957
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(3022..4440)
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
ORIGIN
        1 cgggcctaaa gaactaaaaa atctatctgt ttcttttcat tctctgtatt ttttatagtt
       61 tctgttgcat gggcataaag ttgcaagctt gatatcttga cagctagctc agtcctaggt
      121 ataatactag tcgagaccat tggtctcagt tttagagcta gaaatagcaa gttaaaataa
      181 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgatatcgaa
      241 ttcctgcagc ccgggggatc cactagttct agagcggccg ccaccgcggt ggagctcatc
      301 ccaatggcgc gccgagcttg gctcgagcat ggtcatagct gtttcctgtg tgaaattgtt
      361 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
      421 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
      481 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
      541 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
      601 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      661 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      721 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      781 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      841 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      901 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      961 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1021 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     1081 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     1141 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     1201 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     1261 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     1321 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     1381 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     1441 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttagaaaa
     1501 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1561 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1621 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     1681 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     1741 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     1801 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     1861 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     1921 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     1981 atacctggaa tgctgttttc ccagggatcg cagtggtgag taaccatgca tcatcaggag
     2041 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2101 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2161 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     2221 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     2281 aagacgtttc ccgttgaata tggctcatac tcttcctttt tcaatattat tgaagcattt
     2341 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     2401 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     2461 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg
     2521 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     2581 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     2641 ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt
     2701 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca
     2761 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
     2821 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
     2881 gacgttgtaa aacgacggcc agtgaattga cgcgtattgg gatggtaccg ggccccccct
     2941 cgaggtcgac ggtatcgata cggcatcaga gcagattgta ctgagagtgc accataatcg
     3001 gcattttctt ttgcgttttt atttgttaac tgttaattgt ccttgttcaa ggatgctgtc
     3061 tttgacaaca gatgttttct tgcctttgat gttcagcagg aagctaggcg caaacgttga
     3121 ttgtttgtct gcgtagaatc ctctgtttgt catatagctt gtaatcacga cattgtttcc
     3181 tttcgcttga ggtacagcga agtgtgagta agtaaaggtt acatcgttag gatcaagatc
     3241 catttttaac acaaggccag ttttgttcag cggcttgtat gggccagtta aagaattaga
     3301 aacataacca agcatgtaaa tatcgttaga cgtaatgccg tcaatcgtca tttttgatcc
     3361 gcgggagtca gtgaacagat accatttgcc gttcatttta aagacgttcg cgcgttcaat
     3421 ttcatctgtt actgtgttag atgcaatcag cggtttcatc acttttttca gtgtgtaatc
     3481 atcgtttagc tcaatcatac cgagagcgcc gtttgctaac tcagccgtgc gttttttatc
     3541 gctttgcaga agtttttgac tttcttgacg gaagaatgat gtgcttttgc catagtatgc
     3601 tttgttaaat aaagattctt cgccttggta gccatcttca gttccagtgt ttgcttcaaa
     3661 tactaagtat ttgtggcctt tatcttctac gtagtgagga tctctcagcg tatggttgtc
     3721 gcctgagctg tagttgcctt catcgatgaa ctgctgtaca ttttgatacg tttttccgtc
     3781 accgtcaaag attgatttat aatcctctac accgttgatg ttcaaagagc tgtctgatgc
     3841 tgatacgtta acttgtgcag ttgtcagtgt ttgtttgccg taatgtttac cggagaaatc
     3901 agtgtagaat aaacggattt ttccgtcaga tgtaaatgtg gctgaacctg accattcttg
     3961 tgtttggtct tttaggatag aatcatttgc atcgaatttg tcgctgtctt taaagacgcg
     4021 gccagcgttt ttccagctgt caatagaagt ttcgccgact ttttgataga acatgtaaat
     4081 cgatgtgtca tccgcatttt taggatctcc ggctaatgca aagacgatgt ggtagccgtg
     4141 atagtttgcg acagtgccgt cagcgttttg taatggccag ctgtcccaaa cgtccaggcc
     4201 ttttgcagaa gagatatttt taattgtgga cgaatcgaac tcaggaactt gatatttttc
     4261 atttttttgc tgttcaggga tttgcagcat atcatggcgt gtaatatggg aaatgccgta
     4321 tgtttcctta tatggctttt ggttcgtttc tttcgcaaac gcttgagttg cgcctcctgc
     4381 cagcagtgcg gtagtaaagg ttaatactgt tgcttgtttt gcaaactttt tgatgttcat
     4441 cgttcatgtc tcctttttta tgtactgtgt tagcggtctg cttcttccag ccctcctgtt
     4501 tgaagatggc aagttagtta cgcacaataa aaaaagacct aaaatatgta aggggtgacg
     4561 ccaaagtata cactttgccc tttacacatt ttaggtcttg cctgctttat cagtaacaaa
     4621 cccgcgcgat ttacttttcg acctcattct attagactct cgtttggatt gcaactggtc
     4681 tattttcctc ttttgtttga tagaaaatca taaaaggatt tgcagacta
//
