LOCUS       sequence-428749-        6057 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 6057)
  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 6057)
  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-TP
FEATURES             Location/Qualifiers
     source          1..6057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(21..42)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     233..252
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     465..487
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     479..496
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     480..496
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     503..522
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        503..521
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     551..570
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             complement(join(609..1943,1944..2030))
                     /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      2467..2476
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        join(2492..2497,2498..2514,2515..2521)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     misc_RNA        2544..2619
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2699..2717)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2700..2717)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2735..2751)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2735..2751)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2748..2770)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2759..2775
                     /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(2783..2789,2790..2807,2808..2813))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2828..2849
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             3093..3329
                     /codon_start=1
                     /gene="dhfrIIb from plasmid R388"
                     /product="E. coli plasmid-associated dihydrofolate 
                     reductase"
                     /label=TpR
                     /note="confers resistance to trimethoprim"
                     /translation="MGQSSDEANAPVAGQFALPLSATFGLGDRVRKKSGAAWQGQVVGW
                     YCTKLTPEGYAVESESHPGSVQIYPVAALERVA"
     rep_origin      3431..3782
                     /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      5086..5674
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5575..5594
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5828..5845
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5941..6057,1..339))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
       61 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
      121 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
      181 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
      241 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
      301 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc cattcgccat tcaggctgcg
      361 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      421 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      481 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattcg agctcggtac
      541 ccggggatcc cggcatcaga gcagattgta ctgagagtgc accataatcg gcattttctt
      601 ttgcgttttt atttgttaac tgttaattgt ccttgttcaa ggatgctgtc tttgacaaca
      661 gatgttttct tgcctttgat gttcagcagg aagctaggcg caaacgttga ttgtttgtct
      721 gcgtagaatc ctctgtttgt catatagctt gtaatcacga cattgtttcc tttcgcttga
      781 ggtacagcga agtgtgagta agtaaaggtt acatcgttag gatcaagatc catttttaac
      841 acaaggccag ttttgttcag cggcttgtat gggccagtta aagaattaga aacataacca
      901 agcatgtaaa tatcgttaga cgtaatgccg tcaatcgtca tttttgatcc gcgggagtca
      961 gtgaacagat accatttgcc gttcatttta aagacgttcg cgcgttcaat ttcatctgtt
     1021 actgtgttag atgcaatcag cggtttcatc acttttttca gtgtgtaatc atcgtttagc
     1081 tcaatcatac cgagagcgcc gtttgctaac tcagccgtgc gttttttatc gctttgcaga
     1141 agtttttgac tttcttgacg gaagaatgat gtgcttttgc catagtatgc tttgttaaat
     1201 aaagattctt cgccttggta gccatcttca gttccagtgt ttgcttcaaa tactaagtat
     1261 ttgtggcctt tatcttctac gtagtgagga tctctcagcg tatggttgtc gcctgagctg
     1321 tagttgcctt catcgatgaa ctgctgtaca ttttgatacg tttttccgtc accgtcaaag
     1381 attgatttat aatcctctac accgttgatg ttcaaagagc tgtctgatgc tgatacgtta
     1441 acttgtgcag ttgtcagtgt ttgtttgccg taatgtttac cggagaaatc agtgtagaat
     1501 aaacggattt ttccgtcaga tgtaaatgtg gctgaacctg accattcttg tgtttggtct
     1561 tttaggatag aatcatttgc atcgaatttg tcgctgtctt taaagacgcg gccagcgttt
     1621 ttccagctgt caatagaagt ttcgccgact ttttgataga acatgtaaat cgatgtgtca
     1681 tccgcatttt taggatctcc ggctaatgca aagacgatgt ggtagccgtg atagtttgcg
     1741 acagtgccgt cagcgttttg taatggccag ctgtcccaaa cgtccaggcc ttttgcagaa
     1801 gagatatttt taattgtgga cgaatcgaac tcaggaactt gatatttttc atttttttgc
     1861 tgttcaggga tttgcagcat atcatggcgt gtaatatggg aaatgccgta tgtttcctta
     1921 tatggctttt ggttcgtttc tttcgcaaac gcttgagttg cgcctcctgc cagcagtgcg
     1981 gtagtaaagg ttaatactgt tgcttgtttt gcaaactttt tgatgttcat cgttcatgtc
     2041 tcctttttta tgtactgtgt tagcggtctg cttcttccag ccctcctgtt tgaagatggc
     2101 aagttagtta cgcacaataa aaaaagacct aaaatatgta aggggtgacg ccaaagtata
     2161 cactttgccc tttacacatt ttaggtcttg cctgctttat cagtaacaaa cccgcgcgat
     2221 ttacttttcg acctcattct attagactct cgtttggatt gcaactggtc tattttcctc
     2281 ttttgtttga tagaaaatca taaaaggatt tgcagactac gggcctaaag aactaaaaaa
     2341 tctatctgtt tcttttcatt ctctgtattt tttatagttt ctgttgcatg ggcataaagt
     2401 tgcgtcgacg ccttgctcaa attggaatca ggtttgtgcc aataccagta gaaacagacg
     2461 aagaatccat ggcggccgct gtcatgggcc cttgacaatt aatcatccgg ctcgtataat
     2521 gtgtggcgag accattggtc tcagttttag agctagaaat agcaagttaa aataaggcta
     2581 gtccgttatc aacttgaaaa agtggcaccg agtcggtgct ttttttgaga tctgtccata
     2641 cccatggtct agaatgctcg agacttttca tactcccgcc attcagaagc ttgagtattc
     2701 tatagtgtca cctaaatagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     2761 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     2821 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     2881 cgggaaacct gtcgtgccag aattcttgaa gacgaaaggc cgtgggtcga tgtttgatgt
     2941 tatggagcag caacgatgtt acgcagcagg gcagtcgccc taaaacaatg ttaggcagcc
     3001 gttgtgctgg tgctttctga tagttgttgt ggggtaggca gtcagagttc gatttgcttg
     3061 tcgccataat agattcacaa gaaggattcg acatgggtca aagtagcgat gaagccaacg
     3121 ctcccgttgc agggcagttt gcgcttcccc tgagtgccac ctttggctta ggggatcgcg
     3181 tacgcaagaa atctggtgcc gcttggcagg gtcaagtcgt cggttggtat tgcacaaaac
     3241 tcactcctga aggctatgcg gtcgagtccg aatcccaccc aggctcagtg caaatttatc
     3301 ctgtggctgc acttgaacgt gtggcctaac aattcgctcc agaccggtct tagcccctcc
     3361 ccttggtgtc caaccgctct gtaggcctct caggcgccgc tggtgccgct ggttggacgc
     3421 caagggtgaa tccgcctcga taccctgatt actcgcttcc tgcgccctct caggcggcga
     3481 taggggactg gtaaaacggg gattgcccag acgcctcccc cgccccttca ggggcacaaa
     3541 tgcggcccca acggggccac gtagtggtgc gttttttgcg tttccaccct tttcttcctt
     3601 ttccctttta aaccttttag gacgtctaca ggccacgtaa tccgtggcct gtagagttta
     3661 aaaagggacg gatttgttgc cattaaggga cggatttgtt gttaagaagg gacggatttg
     3721 ttgttgtaaa gggacggatt tgttgtattg tgggacgcag atacagtgtc cccttataca
     3781 caaggaatgt cgaacgtggc ctcaccccca atggtttaca aaagcaatgc cctggtcgag
     3841 gccgcgtatc gcctcagtgt tcaggaacag cggatcgttc tggcctgtat tagccaggtg
     3901 aagaggagcg agcctgtcac cgatgaagtg atgtattcag tgacggcgga ggacatagcg
     3961 acgatggcgg gtgtccctat cgaatcttcc tacaaccagc tcaaagaagc ggccctgcgc
     4021 ctgaaacggc gggaagtccg gttaacccaa gagcccaatg gcaaggggaa aagaccgagt
     4081 gtgatgatta ccggctgggt gcaaacaatc atctaccggg agggtgaggg ccgtgtagaa
     4141 ctcaggttca ccaaagacat gctgccgtac ctgacggaac tcaccaaaca gttcaccaaa
     4201 tacgccttgg ctgacgtggc caagatggac agcacccacg cgatcaggct ttacgagctg
     4261 ctcatgcaat gggacagcat cggccagcgc gaaatagaaa ttgaccagct gcgaaagtgg
     4321 tttcaactgg aaggccggta tccctcgatc aaggacttca agttgcgagt gcttgatcca
     4381 gccgtgacgc agatcaacga gcacagcccg ctacaggtgg agtgggcgca gcgaaagacc
     4441 gggcgcaagg tcacacatct gttgttcagt tttggaccga agaagcccgc caaggcggtg
     4501 ggtaaggccc cagcgaagcg caaggccggg aagatttcag atgctgagat cgcgaaacag
     4561 gctcgccctg gtgagacatg ggaagcggcc cgcgctcgac taacccagat gccgcttgcg
     4621 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     4681 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     4741 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     4801 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     4861 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     4921 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     4981 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5041 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5101 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5161 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5221 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     5281 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     5341 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     5401 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     5461 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     5521 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     5581 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     5641 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     5701 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     5761 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     5821 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct
     5881 tacgcatctg tgcggtattt cacaccgcac agatgcgtaa ggagaaaata ccgcatcagg
     5941 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     6001 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccg
//
