LOCUS       sequence-496372-        6945 bp    DNA     circular SYN 07-JAN-2026
DEFINITION  A sgRNA expression plasmid for genome editing in Pseudomonas.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6945)
  AUTHORS   Chi SI, Akuma M, Xu R, Plante V, Hadinezhad M, Tambong JT
  TITLE     Phenazines are involved in the antagonism of a novel subspecies of 
            Pseudomonas chlororaphis strain S1Bt23 against Pythium ultimum.
  JOURNAL   Sci Rep. 2024 Sep 3;14(1):20517. doi: 10.1038/s41598-024-71418-y.
  PUBMED    39227476
REFERENCE   2  (bases 1 to 6945)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 7, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2024-09-3"; volume: "14(1):20517. doi"; pages: " 
            10.1038/s41598-024-71418-y"
COMMENT     Sequence Label: pAKanCRISPR
FEATURES             Location/Qualifiers
     source          1..6945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(16..37)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     228..247
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     460..482
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     474..491
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     475..491
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     498..517
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        498..516
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     546..565
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(604..1938,1939..2025))
                     /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"
     regulatory      2462..2471
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        join(2487..2492,2493..2509,2510..2516)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     misc_RNA        2539..2614
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2694..2712)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2695..2712)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2730..2746)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2730..2746)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2743..2765)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2754..2770
                     /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(2778..2784,2785..2802,2803..2808))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2823..2844
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2915..2933)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             3182..3976
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3236..3255)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3846..3865
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      4314..4665
                     /label=pRO1600 oriV
                     /note="broad-host-range origin of replication from 
                     Pseudomonas aeruginosa plasmid pRO1600; requires the 
                     pRO1600 Rep protein for replication (West et al., 1994)"
     rep_origin      5969..6557
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6458..6477
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6711..6728
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6824..6945,1..334))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
       61 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
      121 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
      181 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
      241 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
      301 cccgccgcgc ttaatgcgcc gctacagggc gcgtccattc gccattcagg ctgcgcaact
      361 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
      421 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
      481 cgacggccag tgaattgtaa tacgactcac tatagggcga attcgagctc ggtacccggg
      541 gatcccggca tcagagcaga ttgtactgag agtgcaccat aatcggcatt ttcttttgcg
      601 tttttatttg ttaactgtta attgtccttg ttcaaggatg ctgtctttga caacagatgt
      661 tttcttgcct ttgatgttca gcaggaagct aggcgcaaac gttgattgtt tgtctgcgta
      721 gaatcctctg tttgtcatat agcttgtaat cacgacattg tttcctttcg cttgaggtac
      781 agcgaagtgt gagtaagtaa aggttacatc gttaggatca agatccattt ttaacacaag
      841 gccagttttg ttcagcggct tgtatgggcc agttaaagaa ttagaaacat aaccaagcat
      901 gtaaatatcg ttagacgtaa tgccgtcaat cgtcattttt gatccgcggg agtcagtgaa
      961 cagataccat ttgccgttca ttttaaagac gttcgcgcgt tcaatttcat ctgttactgt
     1021 gttagatgca atcagcggtt tcatcacttt tttcagtgtg taatcatcgt ttagctcaat
     1081 cataccgaga gcgccgtttg ctaactcagc cgtgcgtttt ttatcgcttt gcagaagttt
     1141 ttgactttct tgacggaaga atgatgtgct tttgccatag tatgctttgt taaataaaga
     1201 ttcttcgcct tggtagccat cttcagttcc agtgtttgct tcaaatacta agtatttgtg
     1261 gcctttatct tctacgtagt gaggatctct cagcgtatgg ttgtcgcctg agctgtagtt
     1321 gccttcatcg atgaactgct gtacattttg atacgttttt ccgtcaccgt caaagattga
     1381 tttataatcc tctacaccgt tgatgttcaa agagctgtct gatgctgata cgttaacttg
     1441 tgcagttgtc agtgtttgtt tgccgtaatg tttaccggag aaatcagtgt agaataaacg
     1501 gatttttccg tcagatgtaa atgtggctga acctgaccat tcttgtgttt ggtcttttag
     1561 gatagaatca tttgcatcga atttgtcgct gtctttaaag acgcggccag cgtttttcca
     1621 gctgtcaata gaagtttcgc cgactttttg atagaacatg taaatcgatg tgtcatccgc
     1681 atttttagga tctccggcta atgcaaagac gatgtggtag ccgtgatagt ttgcgacagt
     1741 gccgtcagcg ttttgtaatg gccagctgtc ccaaacgtcc aggccttttg cagaagagat
     1801 atttttaatt gtggacgaat cgaactcagg aacttgatat ttttcatttt tttgctgttc
     1861 agggatttgc agcatatcat ggcgtgtaat atgggaaatg ccgtatgttt ccttatatgg
     1921 cttttggttc gtttctttcg caaacgcttg agttgcgcct cctgccagca gtgcggtagt
     1981 aaaggttaat actgttgctt gttttgcaaa ctttttgatg ttcatcgttc atgtctcctt
     2041 ttttatgtac tgtgttagcg gtctgcttct tccagccctc ctgtttgaag atggcaagtt
     2101 agttacgcac aataaaaaaa gacctaaaat atgtaagggg tgacgccaaa gtatacactt
     2161 tgccctttac acattttagg tcttgcctgc tttatcagta acaaacccgc gcgatttact
     2221 tttcgacctc attctattag actctcgttt ggattgcaac tggtctattt tcctcttttg
     2281 tttgatagaa aatcataaaa ggatttgcag actacgggcc taaagaacta aaaaatctat
     2341 ctgtttcttt tcattctctg tattttttat agtttctgtt gcatgggcat aaagttgcgt
     2401 cgacgccttg ctcaaattgg aatcaggttt gtgccaatac cagtagaaac agacgaagaa
     2461 tccatggcgg ccgctgtcat gggcccttga caattaatca tccggctcgt ataatgtgtg
     2521 gcgagaccat tggtctcagt tttagagcta gaaatagcaa gttaaaataa ggctagtccg
     2581 ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgagatctgt ccatacccat
     2641 ggtctagaat gctcgagact tttcatactc ccgccattca gaagcttgag tattctatag
     2701 tgtcacctaa atagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2761 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     2821 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     2881 aacctgtcgt gccagaattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     2941 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     3001 atggcgatag ctagactggg cggttttatg gacagcaagc gaaccggaat tgccagctgg
     3061 ggcgccctct ggtaaggttg ggaagccctg caaagtaaac tggatggctt tcttgccgcc
     3121 aaggatctga tggcgcaggg gatcaagatc tgatcaagag acaggatgag gatcgtttcg
     3181 catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt
     3241 cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc
     3301 agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact
     3361 gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt
     3421 gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca
     3481 ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat
     3541 gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg
     3601 catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga
     3661 agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga
     3721 cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa
     3781 tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga
     3841 catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt
     3901 cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct
     3961 tgacgagttc ttctgactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4021 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4081 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccaattaca cgccactggc
     4141 tgtgcttgct ggggtgacgg tggcaacggt ggcggccttg ctgggctatc gcgttggaaa
     4201 gaaacgaggg aaaggggact gataaaccgg tcttagcccc tccccttggt gtccaaccgc
     4261 tctgtaggcc tctcaggcgc cgctggtgcc gctggttgga cgccaagggt gaatccgcct
     4321 cgataccctg attactcgct tcctgcgccc tctcaggcgg cgatagggga ctggtaaaac
     4381 ggggattgcc cagacgcctc ccccgcccct tcaggggcac aaatgcggcc ccaacggggc
     4441 cacgtagtgg tgcgtttttt gcgtttccac ccttttcttc cttttccctt ttaaaccttt
     4501 taggacgtct acaggccacg taatccgtgg cctgtagagt ttaaaaaggg acggatttgt
     4561 tgccattaag ggacggattt gttgttaaga agggacggat ttgttgttgt aaagggacgg
     4621 atttgttgta ttgtgggacg cagatacagt gtccccttat acacaaggaa tgtcgaacgt
     4681 ggcctcaccc ccaatggttt acaaaagcaa tgccctggtc gaggccgcgt atcgcctcag
     4741 tgttcaggaa cagcggatcg ttctggcctg tattagccag gtgaagagga gcgagcctgt
     4801 caccgatgaa gtgatgtatt cagtgacggc ggaggacata gcgacgatgg cgggtgtccc
     4861 tatcgaatct tcctacaacc agctcaaaga agcggccctg cgcctgaaac ggcgggaagt
     4921 ccggttaacc caagagccca atggcaaggg gaaaagaccg agtgtgatga ttaccggctg
     4981 ggtgcaaaca atcatctacc gggagggtga gggccgtgta gaactcaggt tcaccaaaga
     5041 catgctgccg tacctgacgg aactcaccaa acagttcacc aaatacgcct tggctgacgt
     5101 ggccaagatg gacagcaccc acgcgatcag gctttacgag ctgctcatgc aatgggacag
     5161 catcggccag cgcgaaatag aaattgacca gctgcgaaag tggtttcaac tggaaggccg
     5221 gtatccctcg atcaaggact tcaagttgcg agtgcttgat ccagccgtga cgcagatcaa
     5281 cgagcacagc ccgctacagg tggagtgggc gcagcgaaag accgggcgca aggtcacaca
     5341 tctgttgttc agttttggac cgaagaagcc cgccaaggcg gtgggtaagg ccccagcgaa
     5401 gcgcaaggcc gggaagattt cagatgctga gatcgcgaaa caggctcgcc ctggtgagac
     5461 atgggaagcg gcccgcgctc gactaaccca gatgccgctt gcgcaaacta ttaactggcg
     5521 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     5581 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     5641 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     5701 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     5761 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     5821 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5881 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5941 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6001 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6061 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     6121 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6181 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6241 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6301 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6361 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6421 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6481 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6541 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6601 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6661 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     6721 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     6781 tttcacaccg cacagatgcg taaggagaaa ataccgcatc aggaaattgt aagcgttaat
     6841 attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc
     6901 gaaatcggca aaatccctta taaatcaaaa gaatagaccg agata
//
