LOCUS       sequence-428747-        6539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A sgRNA expression plasmid for genome editing in Burkholderia.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6539)
  AUTHORS   Ferreira MR, Queiroga V, Moreira LM
  TITLE     Genomic editing in Burkholderia multivorans by CRISPR/Cas9.
  JOURNAL   Appl Environ Microbiol. 2024 Feb 21;90(2):e0225023. doi: 
            10.1128/aem.02250-23. Epub 2024 Feb 2.
  PUBMED    38299816
REFERENCE   2  (bases 1 to 6539)
  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."; date: "2024-02-21"; volume: "90(2):e0225023. 
            doi"; pages: " 10.1128/aem.02250-23. Epub 2024 Feb 2"
COMMENT     Sequence Label: pACRISPR-CM
FEATURES             Location/Qualifiers
     source          1..6539
                     /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."
     promoter        3021..3123
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(3190..3209)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     rep_origin      3908..4259
                     /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      5563..6151
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6052..6071
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6305..6322
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(6418..6539,1..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 aaggtcgaat aaatacctgt gacggaagat
     2941 cacttcgcag aataaataaa tcctggtgtc cctgttgata ccgggaagcc ctgggccaac
     3001 ttttggcgaa aatgagacgt tgatcggcac gtaagaggtt ccaactttca ccataatgaa
     3061 ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct
     3121 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa
     3181 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg
     3241 gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt
     3301 attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac
     3361 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact
     3421 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata
     3481 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt
     3541 gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac
     3601 gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa
     3661 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc
     3721 catgtcggca gaatgcttaa tgaattacaa cagtttttat gcatgcgccc aatacgcaaa
     3781 ccgcctctcc ccgcgcgttg gccgattcat taatgcagca ccggtcttag cccctcccct
     3841 tggtgtccaa ccgctctgta ggcctctcag gcgccgctgg tgccgctggt tggacgccaa
     3901 gggtgaatcc gcctcgatac cctgattact cgcttcctgc gccctctcag gcggcgatag
     3961 gggactggta aaacggggat tgcccagacg cctcccccgc cccttcaggg gcacaaatgc
     4021 ggccccaacg gggccacgta gtggtgcgtt ttttgcgttt ccaccctttt cttccttttc
     4081 ccttttaaac cttttaggac gtctacaggc cacgtaatcc gtggcctgta gagtttaaaa
     4141 agggacggat ttgttgccat taagggacgg atttgttgtt aagaagggac ggatttgttg
     4201 ttgtaaaggg acggatttgt tgtattgtgg gacgcagata cagtgtcccc ttatacacaa
     4261 ggaatgtcga acgtggcctc acccccaatg gtttacaaaa gcaatgccct ggtcgaggcc
     4321 gcgtatcgcc tcagtgttca ggaacagcgg atcgttctgg cctgtattag ccaggtgaag
     4381 aggagcgagc ctgtcaccga tgaagtgatg tattcagtga cggcggagga catagcgacg
     4441 atggcgggtg tccctatcga atcttcctac aaccagctca aagaagcggc cctgcgcctg
     4501 aaacggcggg aagtccggtt aacccaagag cccaatggca aggggaaaag accgagtgtg
     4561 atgattaccg gctgggtgca aacaatcatc taccgggagg gtgagggccg tgtagaactc
     4621 aggttcacca aagacatgct gccgtacctg acggaactca ccaaacagtt caccaaatac
     4681 gccttggctg acgtggccaa gatggacagc acccacgcga tcaggcttta cgagctgctc
     4741 atgcaatggg acagcatcgg ccagcgcgaa atagaaattg accagctgcg aaagtggttt
     4801 caactggaag gccggtatcc ctcgatcaag gacttcaagt tgcgagtgct tgatccagcc
     4861 gtgacgcaga tcaacgagca cagcccgcta caggtggagt gggcgcagcg aaagaccggg
     4921 cgcaaggtca cacatctgtt gttcagtttt ggaccgaaga agcccgccaa ggcggtgggt
     4981 aaggccccag cgaagcgcaa ggccgggaag atttcagatg ctgagatcgc gaaacaggct
     5041 cgccctggtg agacatggga agcggcccgc gctcgactaa cccagatgcc gcttgcgcaa
     5101 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5161 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5221 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5281 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5341 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5401 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5461 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5521 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5581 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5641 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5701 aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5761 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5821 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5881 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5941 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6001 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6061 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6121 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6181 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6241 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     6301 gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg atgcggtatt ttctccttac
     6361 gcatctgtgc ggtatttcac accgcacaga tgcgtaagga gaaaataccg catcaggaaa
     6421 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     6481 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagata
//
