LOCUS       Exported                4729 bp ds-DNA     circular SYN 12-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
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4729
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     432..448
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     433..449
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(511..1932)
                     /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        2318..2352
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     misc_RNA        2370..2445
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2478..2494)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2478..2494)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2553..2569)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2553..2569)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2566..2588)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2577..2593
                     /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(2601..2631)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2646..2667
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2784..2801)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2955..3543)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3035..3054)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3714..4529)
                     /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     4437..4456
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(4530..4634)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4702..4720
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     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 ccagtgaatt gacgcgtatt gggatggtac
      421 cgggcccccc ctcgaggtcg acggtatcga tacggcatca gagcagattg tactgagagt
      481 gcaccataat cggcattttc ttttgcgttt ttatttgtta actgttaatt gtccttgttc
      541 aaggatgctg tctttgacaa cagatgtttt cttgcctttg atgttcagca ggaagctagg
      601 cgcaaacgtt gattgtttgt ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac
      661 gacattgttt cctttcgctt gaggtacagc gaagtgtgag taagtaaagg ttacatcgtt
      721 aggatcaaga tccattttta acacaaggcc agttttgttc agcggcttgt atgggccagt
      781 taaagaatta gaaacataac caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt
      841 catttttgat ccgcgggagt cagtgaacag ataccatttg ccgttcattt taaagacgtt
      901 cgcgcgttca atttcatctg ttactgtgtt agatgcaatc agcggtttca tcactttttt
      961 cagtgtgtaa tcatcgttta gctcaatcat accgagagcg ccgtttgcta actcagccgt
     1021 gcgtttttta tcgctttgca gaagtttttg actttcttga cggaagaatg atgtgctttt
     1081 gccatagtat gctttgttaa ataaagattc ttcgccttgg tagccatctt cagttccagt
     1141 gtttgcttca aatactaagt atttgtggcc tttatcttct acgtagtgag gatctctcag
     1201 cgtatggttg tcgcctgagc tgtagttgcc ttcatcgatg aactgctgta cattttgata
     1261 cgtttttccg tcaccgtcaa agattgattt ataatcctct acaccgttga tgttcaaaga
     1321 gctgtctgat gctgatacgt taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt
     1381 accggagaaa tcagtgtaga ataaacggat ttttccgtca gatgtaaatg tggctgaacc
     1441 tgaccattct tgtgtttggt cttttaggat agaatcattt gcatcgaatt tgtcgctgtc
     1501 tttaaagacg cggccagcgt ttttccagct gtcaatagaa gtttcgccga ctttttgata
     1561 gaacatgtaa atcgatgtgt catccgcatt tttaggatct ccggctaatg caaagacgat
     1621 gtggtagccg tgatagtttg cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca
     1681 aacgtccagg ccttttgcag aagagatatt tttaattgtg gacgaatcga actcaggaac
     1741 ttgatatttt tcattttttt gctgttcagg gatttgcagc atatcatggc gtgtaatatg
     1801 ggaaatgccg tatgtttcct tatatggctt ttggttcgtt tctttcgcaa acgcttgagt
     1861 tgcgcctcct gccagcagtg cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt
     1921 tttgatgttc atcgttcatg tctccttttt tatgtactgt gttagcggtc tgcttcttcc
     1981 agccctcctg tttgaagatg gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg
     2041 taaggggtga cgccaaagta tacactttgc cctttacaca ttttaggtct tgcctgcttt
     2101 atcagtaaca aacccgcgcg atttactttt cgacctcatt ctattagact ctcgtttgga
     2161 ttgcaactgg tctattttcc tcttttgttt gatagaaaat cataaaagga tttgcagact
     2221 acgggcctaa agaactaaaa aatctatctg tttcttttca ttctctgtat tttttatagt
     2281 ttctgttgca tgggcataaa gttgcaagct tgatatcttg acagctagct cagtcctagg
     2341 tataatacta gtcgagacca ttggtctcag ttttagagct agaaatagca agttaaaata
     2401 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgatatcga
     2461 attcctgcag cccgggggat ccactagttc tagagcggcc gccaccgcgg tggagctcat
     2521 cccaatggcg cgccgagctt ggctcgagca tggtcatagc tgtttcctgt gtgaaattgt
     2581 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     2641 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2701 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2761 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2821 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2881 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2941 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     3001 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     3061 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     3121 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     3181 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3241 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3301 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3361 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     3421 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3481 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3541 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3601 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3661 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttagaaa
     3721 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     3781 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     3841 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     3901 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     3961 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     4021 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     4081 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     4141 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     4201 aatacctgga atgctgtttt cccagggatc gcagtggtga gtaaccatgc atcatcagga
     4261 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     4321 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     4381 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     4441 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     4501 caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta ttgaagcatt
     4561 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4621 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     4681 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtc
//
